In vitro mutagenesis, phenotyping, and gene mapping

Description of equivalent WO 03093426 A2

IN VITRO MUTAGENESIS, PHENOTYPING, AND GENE MAPPING

Cross Reference to Related Applications This application is based on and claims priority to United States Provisional Application Serial Number 60/377,864, filed May 2, 2002, herein incorporated by reference in its entirety.

Technical Field The presently claimed subject matter pertains to novel cellular libraries useful for phenotypic screening and gene mapping.

Table of Abbreviations

6-MP 6-mercaptopurine

6-TG 6-thioguanine

6-TX 6-thioxanthine

AA acrylamide monomer att Int recombination site attB Int recombination site attP Int recombination site β-geo β -galactosidase / neomycin fusion

CAD carbamoyl-phosphate synthetase 2/ aspartate transcarbamylase/ dihydroorotase

CaMV Cauliflower Mosaic Virus

CFP cyan fluorescent protein

CHL chlorambucil cM centimorgan

CPP cyclophosphamide

CT computerized tomography

DES diethyl sulfate

DMSO dimethylsulfoxide

EC embryonic carcinoma (cell)

EDTA ethylene diamine tetraacetic acid

EG embryonic germ (cell)

EMS ethyl methane sulfonate

ENU Λ.-ethyl-Λ/-nitrosurea ES embryonic stem (cell)

ESI-MS electrospray ionization mass spectrometry

FACS fluorescence-activated cell sorter

FIAU 2'-fluoro-2'-deoxy-1 -β-D- arabinofuranosyl-5-iodo-uracil

FLP FLP recombinase

FLPe enhanced FLP recombinase

FRT FLP recombination site

GC-MS gas chromatography-mass spectrometry

GFP green fluorescent protein

HAT hypoxanthine-aminopterin-thymidine

HBSS Hank's Balanced Salt Solution

His₆ peptide of 6 histidine residues

His₆-NLS-Cre-MTS - cell-permeable Cre recombinases

HPLC high performance liquid chromatography

HPRT hypoxanthine guanine phosphoribosyltransferase

HSVtk herpes simplex virus thymidine kinase

IHF integration host factor

Int integrase recombinases kb kilobase(s)

LC-MS liquid chromatography-mass spectrometry

LDLR low-density lipoprotein receptor

LD-MS laser-desorption mass spectrometry

LIF leukemia inhibitory factor

LNGFR low-affinity nerve growth factor receptor

LOD logarithmic odds ratio

Lox Cre recombination site LoxP wild type lox site

Lox66 mutant lox site

Lox71 mutant lox site

LRS likelihood ratio statistic

M Morgan

MALDI-MS matrix-assisted laser desorption/ ionization mass spectrometry

MALDI-TOF matrix-assisted laser desorption/ ionization - time-of-flight

MCI a synthetic herpes simplex virus thymidine kinase promoter

MLP melphalan

MMC mitomycin C

MMS methyl methane sulfonate

MNNG /V-methyl-Λ/'-nitro-Λ/-

MNU methylnitrosourea

MRI magnetic resonance imaging

MTS membrane translocating sequence

MTT 3-(4,5-dimethylthiazole-2-yl)-2,5- diphenyl tetrazolium bromide

NLS nuclear localization signal

NEO neomycin

NMM Λ/-methyl mesoporphyrin IX

NSM negative selectable marker

PCR polymerase chain reaction

PGK phosphoglycerate kinase I

PMEF primary mouse embryonic fibroblast

PRC procarbazine

PSM positive selectable marker

PURO puromycin

Puro^r puromycin resistance

RecQ a helicase

RFLP restriction fragment length polymorphism RIST - Recombinant Inbred Segregation

Test RLGS - restriction landmark genomic scanning

RS - recombination site

SNP - single nucleotide polymorphism

SSLP - short sequence length polymorphism

STRP - short tandem repeat polymorphism TEM - triethylene melamine tk - thymidine kinase

TOF-MS - time-of-flight mass spectrometry

UR - urethane

UV - ultraviolet XTT - 2,3-Bis(2-methoxy-4-nitro-5- sulfophenyl)-2H-tetrazolium-5- carboxanilide YFP - yellow fluorescent protein

Background Art Complete or draft versions of genome sequences have been obtained in a variety of organisms, including human (Hattori et al., 2000; Lander et al., 2001 ; McPherson et al., 2001 ; Sachidanandam et al., 2001 ; Venter et al., 2001), and a large fraction of the genes are also mapped to chromosomal regions. A current challenge is to annotate gene maps with phenotypic information that imparts functional meaning to the genomic sequences. Thus, genomic research efforts have shifted to systematic determination of gene function (e.g., via analysis of mutant phenotypes). See e.g., Parinov & Sundaresan, 2000; Beckers & Angelis, 2001; Rossant & McKerlie, 2001 ; Yaspo, 2001. The ability to link a phenotype with one or more genes responsible for that trait provides opportunities for new diagnostics and treatments of genetic diseases.

Large-scale random mutagenesis approaches have generally relied on creating or inducing genetic modifications, the impact of which are evaluated in the context of a complete organism. Screening approaches for the selection of both dominant and recessive mutations are available in plant and animal model organisms, including Drosophila melanogaster (Gans et al., 1975; Nusslein- Volhard & Wieschaus, 1980), Caenorhabditis elegans (Brenner, 1974; Kemphues et al., 1988), Arabidopsis thaliana (Budziszewski et al., 2001 ; McElver ef al., 2001 ), Danio rerio (Driever ef al., 1996; Haffter ef al., 1996), and Mus musculus (see citations below). For recovery of mutations that confer early organismal lethality, methods have been developed for the systematic generation of mosaic animals bearing homozygous mutant clones. See e.g., Xu etal., 1995; Duffy etal., 1998. For insights into human disease, the mouse is an experimental genetic system of choice because its genes, biochemical pathways, and physiological organ functions are closely related to those in humans. Random mutagenesis screens in mouse initially focused on screens for dominant mutations that result in viable, clinically relevant phenotypes (Hrabe de Angelis etal., 2000; Isaacs et al., 2000; Nolan et al., 2000). Genome-wide screens that select recessive mutations (Kasarskis et al., 1998; Fahrer et al., 2001) and screens based on mosaic analysis (Liu ef al., 2002) have more recently been undertaken.

Since phenotypic screens have relied on whole organism analysis, systematic mutagenesis studies have been limited to model genetic organisms. Thus, a functional genomics approach has generally not been available in most organisms, including humans, agriculturally important plants and animals, domestic animals, pathogens, etc. However, genomic sequencing has been accomplished or is currently sought in many non-model organisms, and functional annotation is similarly valuable. A reverse genetics strategy called double-stranded RNA interference has been developed recently as a method for functional analysis in non-model organisms. According to this approach, double-stranded RNA is used to target specific RNA transcripts for degradation, thereby leading to a loss of gene function. Since the double-stranded RNA is prepared based on known sequence, the link between gene and phenotype is already known.

RNA interference is a silencing phenomenon that is manifest in plants, animals and fungi, and therefore enables systematic functional analysis of any organism for which genomic sequence data is known. See Zamore, 2001 ; Carthew, 2001. This strategy has been adopted for genome-wide analysis in C. θlegans (Bargmann, 2001). Despite its utility in diverse organisms, RNA interference is limited to loss-of-function analysis. Thus, this strategy is inapplicable for the discovery of disease-related mutations resulting from increased or otherwise altered gene function. Thus, current and long-felt needs in the field include strategies for rapid phenotyping and gene mapping that can be performed in any species. The presently claimed subject matter discloses methods for generating, phenotyping, and mapping mutations in vitro, and thus addresses the current and long-felt need in the art for the same. Summary

The presently claimed subject matter provides cellular libraries useful for in vitro phenotyping and gene mapping, and methods for using the same.

In one embodiment of the presently claimed subject matter, a heterozygous cellular library is provided, the heterozygous cellular library comprising a randomly mutagenized population of isolated cells, wherein each of the isolated cells comprises a marked chromosome comprising a dominant positive selectable marker.

Each cell of a randomly mutagenized population of isolated cells can comprise one or more heterozygous genetic modifications. In one embodiment, the one or more heterozygous genetic modifications are produced by a method selected from the group consisting of chemical mutagenesis, ultraviolet radiation, X-ray radiation, exposure to inhibitors of DNA repair, and combinations thereof.

The presently claimed subject matter also provides methods for preparing a heterozygous cellular library. A representative embodiment of the method comprises: (a) providing a plurality of isolated cells, wherein each ofthe plurality of isolated cells comprises a dominant positive selectable marker; and (b) randomly mutagenizing the plurality of isolated cells, whereby a heterozygous cellular library is prepared. In another embodiment of the presently claimed subject matter, a homozygous cellular library is provided, the homozygous cellular library comprising a randomly mutagenized and homozygosed population of isolated cells. In one embodiment, each cell of a randomly mutagenized and homozygosed population of isolated cells comprises one or more homozygous genetic modifications.

The presently claimed subject matter also provides methods for preparing a homozygous cellular library. A representative embodiment of the method comprises: (a) providing a heterozygous cellular library comprising a plurality of isolated parent cells; (b) inducing site-specific mitotic recombination in the plurality of isolated parent cells; (c) culturing the plurality of isolated parent cells, whereby a population of daughter cells is produced; and (d) selecting daughter cells comprising a homozygous genetic modification, whereby a homozygous cellular library is prepared. In accordance with the disclosed methods for preparing a homozygous cellular library, each of the plurality of isolated parent cells can further comprise a marked chromosome pair, wherein the marked chromosome pair comprises a first chromosome and a second homologous chromosome, wherein the first chromosome comprises a first recombination cassette, and wherein the second homologous chromosome comprises a second allelic recombination cassette. In one embodiment, the first and second recombination cassettes each comprise a centromeric position.

A recombination cassette comprises a recombination site, for example, a lox site, an FRT site, or an att site. In one embodiment, a first recombination cassette and a second recombination cassette can recombine to produce a stable first recombination event. For example, in an exemplary embodiment of the presently claimed subject matter, a first recombination cassette comprises a lox66 site, and a second recombination cassette comprises a lox71 site. In another embodiment of the presently claimed subject matter, a first recombination cassette comprises an attB site, and a second recombination cassette comprises an attP site.

A recombination cassette can further comprise one or more selectable markers to facilitate in vitro phenotyping and gene mapping. For example, a first recombination cassette of a marked chromosome pair can comprise a first selectable marker, and a second recombination cassette of the marked chromosome pair can comprise a second selectable marker. In one embodiment, a selectable marker comprises a dominant negative selectable marker or a recessive positive selectable marker.

A site-specific recombinase used to induce mitotic recombination via the recombination cassettes can comprises a site-specific recombinase selected from the group consisting of a Cre recombinase, a FLP recombinase, and an Int recombinase. A site-specific recombinase comprises in one embodiment a cell- permeable recombinase and in another embodiment a cell-permeable Cre recombinase.

Cells of a cellular library can be isolated from any organism, including both plants and animals. In one embodiment of the presently claimed subject matter, a library of the presently claimed subject matter comprises human cells.

Cells of a cellular library can comprise any suitable cell type, including stem cells, precursor cells, and differentiated cells. In one embodiment of the presently claimed subject matter, a cellular library comprises embryonic stem (ES) cells.

Stem cells and precursor cells of a cellular library can be induced to differentiate in vitro. For example, differentiation can be induced prior to mutagenesis, following mutagenesis and prior to phenotyping, or as part of a phenotyping assay.

In one embodiment, the heterozygous cellular libraries and homozygous cellular libraries of the presently claimed subject matter each comprise a population of genetically related cells. The presently claimed subject matter further provides a kit for in vitro phenotyping and gene mapping. A kit of the presently claimed subject matter comprises: (a) a heterozygous cellular library comprising a randomly mutagenized population of isolated parent cells; and (b) a homozygous cellular library comprising a randomly mutagenized and homozygosed population of isolated daughter cells, wherein the homozygous library is derived from the heterozygous cellular library. In one embodiment, each parent cell of the heterozygous cellular library comprises a unique identifier, and each daughter cell produced by homozygosing a parent cell comprises a same unique identifier. The cellular libraries of the presently claimed subject matter are useful for in vitro phenotyping and gene mapping, as disclosed herein. A phenotype can comprise any observable trait, including but not limited to a visible phenotype, a viability phenotype, a molecular phenotype, a differentiation phenotype, a cell behavioral phenotype, a susceptibility phenotype, a resistance phenotype, and combinations thereof. A genetic locus conferring a phenotype of interest can then be mapped using the disclosed methods.

In a representative embodiment of the presently claimed subject matter, a method for in vitro phenotyping comprises: (a) providing a heterozygous cellular library or a homozygous cellular library; (b) assaying the cellular library for a phenotype of interest; and (c) selecting one or more cells that displays the phenotype of interest.

Representative gene mapping methods provided by the presently claimed subject matter comprise: (a) culturing an isolated cell comprising one or more heterozygous genetic modifications, whereby a population of recombinant cells is produced; and (b) mapping the genomes of individuals within the population of recombinant cells that display a phenotype, whereby a genetic locus that modulates the phenotype is identified. The mapping can comprise, for example, analyzing genetic polymorphisms segregating in the population of recombinant cells.

In accordance with the mapping methods of the presently claimed subject matter, the culturing step can further comprise contacting the culture with an inhibitor of DNA repair to thereby promote mitotic recombination. A representative inhibitor of DNA repair comprises a helicase inhibitor, in one embodiment a RecQ helicase inhibitor. Inhibitor compounds that can be used in accordance with the disclosed methods include, but are not limited to porphyrin and porphyrin derivatives, for example meso-tetra (/V-methyl-4- pyridyl) porphine tetra tosylate (T4) or Λ/-methyl mesoporphyrin IX (NMM).

The presently claimed subject matter further provides a method for in vitro phenotyping and gene mapping comprising: (a) phenotyping a cellular library; (b) selecting a cell comprising a genetic modification that confers a phenotype of interest; (c) culturing a cell that is heterozygous for the genetic modification, whereby a population of recombinant cells is produced; and (d) mapping the genomes of individuals within the population of recombinant cells, whereby a genetic locus that modulates the phenotype is identified. The disclosed method for in vitro phenotyping and gene mapping can employ a heterozygous cellular library or a homozygous cellular library.

Thus, an object of the presently claimed subject matter is to provide novel cellular libraries, and phenotyping and gene mapping methods employing the same. This object has been met in whole or in part by the presently claimed subject matter.

An object of the presently claimed subject matter having been stated hereinabove, other objects will become evident as the description proceeds when taken in connection with the accompanying Drawings and Examples as best described herein below.

Brief Description of the Drawings Figure 1 depicts a summary of the disclosed methods for in vitro phenotyping and gene mapping. Figure 2 is a schematic drawing depicting preparation of a heterozygous cellular library.

Figure 3 is a schematic drawing depicting preparation of a homozygous cellular library. PSM1 , positive selectable marker 1 ; PSM2, positive selectable marker 2; NSM, negative selectable marker; y*****, random mutation; 1A, library position 1A.

Figure 4 is a schematic drawing depicting preparation of a marked chromosome pair as described in Example 1, and preparation of a representative embodiment of a heterozygous cellular library as described in Example 2. Figures 5A-5B are schematic drawings depicting preparation of a representative embodiment of a homozygous cellular library using the heterozygous cellular library of Figure 4. See also Example 4.

Figure 5A is a schematic drawing depicting preparation of a homozygous cellular library using the heterozygous cellular library of Figure 4. Figure 5B is a schematic drawing depicting chromosomal events as cells of the heterozygous library of Figure 4 are expanded in the presence of Cre, 6- TG, puromycin, and neomycin, to thereby produce a homozygous cellular library. Following Cre-induced mitotic recombination, cells comprising a homozygous mutation (circled) are selected based on resistance to puromycin, neomycin, and 6-TG. Puro, puromycin; Neo, neomycin; 6-TG, 6-thioguanine; R, resistant; S, susceptible; y**^*- , random mutation.

Detailed Description J Definitions

While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently claimed subject matter.

Following long-standing patent law convention, the terms "a" and "an" mean "one or more" when used in this application, including the claims. The terms "nucleic acid molecule" and "nucleic acid" each refer to deoxyribonucleotides or ribonucleotides and polymers thereof in single- stranded, double-stranded, or triplexed form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar properties as the reference natural nucleic acid. The terms "nucleic acid molecule" and "nucleic acid" can also be used in place of "gene", "cDNA", or "mRNA."

The term "gene" refers broadly to any segment of DNA associated with a biological function. A gene encompasses sequences including, but not limited to a coding sequence, a promoter region, a cis-regulatory sequence, a non- expressed DNA segment that is a specific recognition sequence for regulatory proteins, a non-expressed DNA segment that contributes to gene expression, a DNA segment designed to have desired parameters, or combinations thereof.

The term "locus" refers to a chromosomal location of a gene.

The term "genotype" refers to the genetic constitution of a cell or organism, e.g. genomic nucleic acid material.

The term "phenotype" generally refers to any observable character of a cell or organism, as described further herein below.

The term "cell" refers to a single cell or a plurality of clonal cells. Thus, the term "cell" encompasses a population of cells produced by clonal expansion of a single cell.

The terms "first" and "second", for example, as used herein to describe homologous chromosomes, recombination cassettes, markers, etc., are included for clarity of description and are not meant to be limiting.

The term "about", as used herein when referring to a measurable value such as a position of a locus (e.g., in cM), target gene strength, power, etc., is meant to encompass variations of in one embodiment ±20% or ±10%, in another embodiment ±5%, in another embodiment ±1%, and in still another embodiment ±0.1 % from the specified value, as such variations are appropriate to perform the disclosed method. ]L Cellular Libraries

The presently claimed subject matter provides cellular libraries and methods for preparing the same. In accordance with the methods disclosed herein, the cellular libraries can be used for in vitro phenotyping and gene mapping, as described herein below. See Figure 1.

The term "cellular library" is used herein to describe a plurality of isolated cells comprising diverse genetic material, wherein each of the plurality of cells comprises one or more random genetic modifications relative to other cells within the cellular library. Each cell of a cellular library is referred to as a "genetic variant."

The term "genetic variant" is used herein to describe genomic nucleic acid material comprising one or more genetic modifications, or to a cell comprising the same. Thus, in one embodiment, each of a plurality of cells of a cellular library prepared as disclosed herein comprises a "genetic variant". A genetic variant can comprise a homozygous or heterozygous genetic modification.

In one embodiment, a cellular library of the presently claimed subject matter comprises a population of genetically related cells. The term "genetically related", as used herein to compare two or more cells, refers to a common cellular origin. Thus, the genomic nucleic acid material of two or more genetically related cells is substantially identical.

The term "substantially identical" refers to genetic identity other than variation produced by: (a) spontaneous mutation; (b) random mutagenesis among genetically identical members of a population; or (c) a combination thereof. Thus, a mutagenized population of initially identical cells are "genetically related. In contrast, the term "genetically related", as used herein to describe a population of cells, excludes a collection of mutants from diverse sources, for example a collection of naturally occurring mutants or a collection of individually targeted mutations. The term "isolated", as used herein to describe a population of cells, refers to a quality of being separate from an organism. For example, the term "isolated cell" refers to a cell that is isolated away from an organism and can be maintained in culture.

The term "culture", as used herein to describe a cell culture, refers to any in vitro setting for cell growth. The term "culture" encompasses the culture of immortal cell lines, primary cell cultures, non-transformed cell lines, and any other cell population that can be maintained in vitro.

In one embodiment, a cellular library of the presently claimed subject matter comprises about 100 members to about 5000 members, or more. A cellular library comprising members having multiple marked chromosome pairs can comprise about 100 members to about 100,000 members, or more. The number of members in a library can be optimized to achieve saturation mutagenesis, wherein a mutant form of each gene is represented. In another embodiment, each cell of a cellular library comprises a unique identifier. In one embodiment of the presently claimed subject matter, a cellular library is spatially arrayed, such that each cell of the cellular library can be identified according to its unique position within the array. For example, a cellular library can be maintained in multi-well plates, and a unique location identity of each cell can be expressed as a plate number and well number.

Cellular libraries of the presently claimed subject matter can be replicated as desired for various applications, including preparing a homozygous cellular library, phenotyping a cellular library, and mapping a genetic locus that confers a phenotype of interest, as described herein below. The term "replicate" refers to the accurate duplication of an original cellular library of the presently claimed subject matter to produce a replica cellular library. Thus, the unique identifier of each cell is identical, or otherwise traceable, in the original and replica libraries.

To facilitate arraying and replicating cellular libraries, automated cell- culturing equipment can be employed, such as the MULTIMEK™96 multi-well pipetter and the BIOMEK®2000 liquid handling workstation (both available from Beckman Instruments, Inc., Fullerton, California, United States of America).

In accordance with the methods of the presently claimed subject matter, cellular libraries can be provided in the form of a kit useful for performing in vitro phenotyping and gene mapping as disclosed herein. For example, a kit can comprise a heterozygous cellular library, a homozygous cellular library, or a combination thereof.

The presently claimed subject matter further provides that cellular libraries can be frozen and transported while frozen to remote locations for performance of in vitro phenotyping and/or gene mapping. Representative methods for cryopreservation of cellular libraries are described in Example 3. Additional methods for preparation and handling of frozen cells can be found in Freshney, 1987 and U.S. Patent Nos. 6,176,089; 6,140,123; 5,629,145; and 4,455,842; among other places.

Heterozygous Cellular Libraries. The term "heterozygous cellular library" refers to a library comprising a randomly mutagenized population of isolated cells. A cell of a heterozygous cellular library comprises a random genetic variant. The term "random genetic variant" refers to a genetic modification induced via random mutagenesis. Random genetic modifications are typically heterozygous, and hence the term "heterozygous cellular library".

In one embodiment, each of the cells of a heterozygous cellular library comprises a marked chromosome comprising a dominant positive selectable marker. The term "marked chromosome" refers to a chromosome comprising a selectable marker that enables identification of cells comprising the marked chromosome in a population of cells, including similar cells that lack the selectable marker. Selection of marker nucleic acids and preparation of marked chromosomes are described further herein below. PCT International Publication No. WO 99/67361 to Wovchik et al. discloses a heterozygous cellular library. In contrast to Wovchik et al., the presently claimed subject matter provides that cells of a heterozygous cellular library can comprise one or more marked chromosomes to facilitate in vitro phenotyping and gene mapping, among other distinctions. The marked chromosomes also facilitate preparation of a homozygous cellular library, which is not readily accomplished in the absence of a marked chromosome.

The presently claimed subject matter further provides a method for preparing a heterozygous cellular library. In a representative embodiment of the presently claimed subject matter, the method comprises: (a) providing a plurality of isolated cells, wherein each of the plurality of isolated cells comprises a dominant positive selectable marker; and (b) randomly mutagenizing the plurality of isolated cells, whereby a heterozygous cellular library is prepared. Representative methods of the presently claimed subject matter are described in Example 2. See also Figures 2 and 4. Homozygous Cellular Libraries. The term "homozygous cellular library" refers to a randomly mutagenized and homozygosed population of isolated cells. Stated another way, a homozygous cellular library comprises a plurality of isolated cells, wherein each of the cells comprises a homozygous genetic variant.

The term "homozygous genetic variant" refers to a cell comprising one or more genetic modifications that are manifested on each allele. Thus, in a diploid organism, a homozygous genetic modification comprises two copies of a homozygous genetic modification. In accordance with the methods of the presently claimed subject matter, a homozygous genetic variant comprises in one embodiment one or more homozygous genetic modifications on a marked chromosome pair, as described herein below.

The term "homozygosed", as used herein to describe a cellular library of the presently claimed subject matter, refers to a population of isolated cells, wherein each cell comprises one or more homozygous genetic variants.

The presently claimed subject matter further provides a method for making a homozygous cellular library via homozygosing of a heterozygous cellular library. The term "homozygosing" refers to a process whereby cells comprising one or more randomly induced genetic modifications are rendered homozygous at loci comprising the one or more randomly induced genetic modifications.

In a representative embodiment of the presently claimed subject matter, the method comprises: (a) providing a heterozygous cellular library comprising a plurality of isolated parent cells; (b) inducing site-specific mitotic recombination in the plurality of isolated parent cells; (c) culturing the plurality of isolated parent cells, whereby a population of daughter cells is produced; and (d) selecting daughter cells comprising a homozygous genetic modification, whereby a homozygous cellular library is prepared. Representative methods for preparing a homozygous cellular library are described in Example 4. See also Figures 3, 5A, and 5B.

The term "parent cell" is used herein to refer to a cell comprising a cell that can undergo mitotic cell division to thereby produce one or more daughter cells. A population or plurality of parent cells can comprise symmetrically dividing parent cells, asymmetrically dividing parent cells, or a combination thereof.

The term "daughter cell" is used herein to refer to any cell that is produced as a result of a mitotic cell division. 11 Cells A cell of a cellular library can comprise any cell type capable of being cultured, i.e., any cell that can undergo mitotic cell division in culture. Thus, a cell of a cellular library comprises two or more homologous chromosomes (e.g., diploid cells, tetraploid cells, etc.).

A significant advantage of the in vitro phenotyping and gene mapping methods that employ cellular libraries of the presently claimed subject matter is the ability to perform such methods using any cell of interest. Prior to the present disclosure, systematic mutagenesis and gene mapping studies have been substantially confined to model genetic organisms. In contrast, the cellular libraries of the presently claimed subject matter can comprise any cell that can be cultured.

Thus, a cell of a cellular library can comprise a plant cell or an animal cell derived from any species including, but not limited to cells derived from pathogens, domestic animals, agriculturally important animals and plants, etc.

In one embodiment of the presently claimed subject matter, cells of a cellular library comprise human cells.

Representative cell types that can be used to prepare a cellular library include but are not limited to differentiated cells and undifferentiated cells, for example stem cells and precursor cells.

The term "differentiated cell" refers to mature cell type. Differentiated cells are typically post-mitotic.

The term "stem cell" refers to a cell that undergoes asymmetric cell division to generate one or more progeny cells and to regenerate itself. Thus, a stem cell is pluripotent in that it can give rise to multiple cell types.

The term "precursor cell" refers to an undifferentiated cell derived from a stem cell, and is not itself a stem cell. A precursor cell can also show pluripotency in that its progeny are capable of differentiating into more than one cell type. A precursor cell does not exhibit self-maintenance. Representative precursor cells include embryonic carcinoma (EC) cells and an embryonic germ (EG) cells. In one embodiment of the presently claimed subject matter, a cell of a cellular library comprises a totipotent stem cell, which can be used to produce chimeras and non-human cloned organisms for additional phenotypic analysis. For example, an exemplary cell comprises an ES cell, which can itself develop into an entire animal. Other exemplary cells are callus cells and/or cells that can be induced to form callus: cells which have the capacity to regenerate as whole plants.

ES cells can be isolated from any suitable source including, but not limited to the inner cell mass of blastocyst stage embryos (Evans & Kaufman, 1981 ; Martin, 1981 ; Magnuson ef al., 1982; Doetschman et al., 1988), disaggregated morulae (Eistetter, 1988), and primordial germ cells (Matsui ef al., 1992; Resnick et al., 1992).

Methods for isolating and culturing ES cells are known to one skilled in the art. Representative protocols for culture of primate ES cells and mouse ES cells can be found, for example, in Joyner, 2000 and in Tymms & Kola, 2001 , among other places. See also U.S. Patent Nos. 6,190,910, and 6,200,806.

ES cells can also be obtained from agriculturally important animals, including chick, cattle, sheep, goats, rabbits, and mink. Representative isolation and culture methods are described, for example, in PCT International Publication Nos. WO 97/20035 and WO 01/11019, and in U.S. Patent No.

6,333,192, among other places.

In one embodiment of the presently claimed subject matter, the screening and mapping methods disclosed herein are performed using cellular libraries comprising human ES cells. Representative human ES cells that can be used in accordance with the methods of the presently claimed subject matter include, but are not limited to those human ES cell lines available from ES Cell International (Melbourne, Australia) and from Wisconsin Alumni Research Foundation (Madison, Wisconsin, United States of America). Additional representative human ES cell lines and methods for culturing the same are available from the NIH Human Embryonic Stem Cell Registry, which can be accessed electronically at http://purl.access.gpo.gov/GPO/LPS15792.

Cellular libraries comprising stem cells and precursor cells can be induced to differentiate in vitro. An ES cell can be induced to differentiate in vitro into extraembryonic lineages, somatic cell lineages, or a combination thereof. ES cells can give rise to derivatives of all three germ layers.

Differentiation can be induced prior to, concurrent with, or following random mutagenesis. In one embodiment of the presently claimed subject matter, stem cells are induced to differentiate prior to performing a phenotype assay as described herein below. Thus, cellular libraries can be prepared comprising cell types relevant to a phenotype to be screened, for example any one of cardiomyocytes, smooth muscle cells, adipocytes, hematopoietic progenitors, yolk sac, skeletal myocytes, chondrocytes, endothelial cells, melanocytes, neurons, glia, pancreatic islet cells, and primitive endoderm. II.B. Marked Chromosomes

The presently claimed subject matter provides cellular libraries comprising a plurality of isolated cells, wherein each of the cells comprises one or more marked chromosomes. As noted herein above, the term "marked chromosome" refers to a chromosome comprising a selectable marker, which is used to select a subpopulation of cells based on expression of the selectable marker, as described further herein below. A selectable marker can be included as part of a recombination cassette, to thereby facilitate selection of a subpopulation following induced mitotic exchange, also described herein below.

In one embodiment of the presently claimed subject matter, each ofthe cells of a cellular library comprises a marked chromosome pair. The term "chromosome pair" as used herein refers to a pair of homologous chromosomes, and further to sets of paired chromosomes. For example, a homologous chromosome pair can comprise a first chromosome and a second homologous chromosome, wherein the first and second chromosomes are each derived from a different parent. A homologous chromosome pair can also comprise a collection of homologous chromosomes present in multiples of two chromosomes (i.e., sets of paired chromosomes), for example as occurs in tetraploid or other polyploid cells.

The term "marked chromosome pair" refers to a homologous chromosome pair wherein each of the homologous chromosomes comprises a selectable marker.

Cells of a cellular library disclosed herein can comprise a single marked chromosome or chromosome pair, or multiple marked chromosomes or chromosome pairs. Marked chromosomes can be prepared by any suitable method for cellular transformation, whereby a heterologous nucleic acid is incorporated into a host cell genome. The term "heterologous nucleic acid" refers to a nucleic acid molecule that originates from a source foreign to an intended host cell or, if from the same source, is modified from its original form. Thus, a heterologous nucleic acid in a host cell includes a gene that is endogenous to the particular host cell but has been modified, for example by mutagenesis or by isolation from native cis-regulatory sequences. The term "heterologous nucleic acid" also includes non-naturally occurring multiple copies of a native nucleic acid. In one embodiment of the presently claimed subject matter, marked chromosomes are prepared by gene targeting. The term "gene targeting" generally refers to methods for targeting a heterologous nucleic acid to a predetermined endogenous target DNA sequence in a cell.

The terms "target DNA sequence" and "target site" are used herein interchangeably to refer to DNA intended to be modified by gene targeting. The target DNA sequence can a gene, or part thereof (e.g., an intron, exon, or regulatory sequence) or an intergenic region.

The term "predetermined" refers to a chromosomal site that is selected at the discretion of the practitioner on the basis of known or predicted sequence information, and it is not constrained to specific sequences recognized by site- specific recombinases, as described further herein below. Essentially any chromosomal site for which sequence data is known can be selected as a predetermined site.

The term "targeting DNA" refers to a nucleic acid molecule comprising: (a) a nucleotide sequence to be introduced into the genome; and (b) a nucleotide sequence homologous to the target site. In accordance with the methods of the presently claimed subject matter, a nucleotide sequence to be introduced into the genome includes a selectable marker and/or a recombination cassette to thereby facilitate the preparation of cellular libraries, phenotyping, and mapping methods disclosed herein.

A nucleotide sequence homologous to the target site can be isogenic with the target site to thereby promote the frequency of homologous recombination as described in U.S. Patent No. 5,789,215.

However, homologous nucleotide sequences that are not isogenic to the target site can also be used. Although it has been demonstrated that the frequency of homologous recombination can be adversely affected by the presence of mismatches between the targeting DNA and the target site, isogenicity is not strictly required. See Sedivy et al., 1999. A nucleotide sequence homologous to the target site is in one embodiment at least about 90% identical to the target site, in another embodiment at least about 95% identical to the target site, and in still another embodiment at least about 99% identical to the target site. Optionally, cellular mismatch repair enzymes can be transitorily inactivated (e.g., by provision of a modulator) to promote recombination of DNA sequences having mismatched bases as described in U.S. Patent No. 5,965,415.

A targeting DNA can be carried in a vector. The term "vector" is used herein to refer to a nucleic acid molecule having nucleotide sequences that enable its replication in a host cell. A vector can also include nucleic acids to permit ligation of nucleotide sequences within the vector, wherein such nucleic acids are also replicated in a host cell. Representative vectors include plasmids, cosmids, and viral vectors. The term "vector" is also used to describe an expression construct, wherein the expression construct comprises a vector and a nucleic acid operatively inserted with the vector, such that the nucleic acid is expressed. Suitable expression vectors that can be used include, but are not limited to, the following vectors or their derivatives: plasmid and cosmid DNA vectors; viruses such as vaccinia virus or adenovirus, baculovirus vectors, yeast vectors, and bacteriophage vectors (e.g., λ phage).

Vectors can also comprise nucleic acids including expression control elements, such as transcription/translation control signals, origins of replication, polyadenylation signals, internal ribosome entry sites, promoters, enhancers, etc., wherein the control elements are operatively associated with a nucleic acid encoding a gene product. Selection of these and other common vector elements are conventional and many such sequences can be derived from commercially available vectors. See e.g., Sambrook & Russell, 2001, and references cited therein.

Representative protocols for the design of gene targeting vectors can be found, for example, in Hasty et al., 2000; Joyner, 2000; and U.S. Patent Nos. 5,789,215; 5,859,307; 6,255,113; and 6,319,692; among other places. Microhomologous recombination in yeast or bacteria, which requires regions of homology as short as 30 base pairs, can be used to facilitate construction of vectors for gene targeting in cells where longer regions of homology to a target site are used for efficient gene targeting. According to this approach, a genomic clone containing the target site in a yeast or bacteria shuttle vector is co-transformed with a targeting DNA into yeast. The targeting DNA can be generated by PCR amplification of the nucleotide sequence to be introduced (e.g., a selectable marker) using primers homologous to the target site. Within the yeast cell, homologous recombination occurs between the targeting DNA and the genomic clone, to thereby produce a targeting DNA comprising longer regions of homology to the target site. See e.g., Zhang etal., 2002; Khrebtukova etal., 1998; and U.S. Patent Nos.6,221, 647 and 6,069,010. A targeting vector can be introduced into targeting cells using any suitable method for introducing DNA into cells, including but not limited to microinjection, electroporation, calcium phosphate precipitation, liposome-mediated delivery, viral infection, protoplast fusion, and particle-mediated uptake. Representative methods relevant to transformation of animal cells can be found in, for example, Capecchi, 1980; Potter et al., 1984; Mannino & Gould-Fogerite, 1988; Slilaty & Aposhian, 1983; Rassoulzadegan ef al., 1982; Armaleo et al., 1990; and Sambrook & Russell, 2001 ; among other places.

Optionally, a targeting DNA is co-administered with a recombinase, for example recA, to a target cell to thereby promote a rate of gene targeting. For example, a recombinase protein(s) can be loaded onto a targeting DNA as described in U.S. Patent No. 6,255,113. To enhance the loading process, a targeting DNA can contain one or more recombinogenic nucleation sequences. A targeting DNA can also be coated with a recombinase protein by pre- incubating the targeting polynucleotide with a recombinase, whereby the recombinase is non-covalently bound to the polynucleotide. See also U.S. Patent No. 5,780,296. Selection of cells comprising a homologous integration event is dependent on the design of the targeting vector employed. Representative methods are described in Hasty etal., 2000; Joyner, 2000; and U.S. Patent Nos.5,789,215; 5,859,307; 6,255,113; and 6,319,692; among other places.

Representative protocols for gene targeting in cells of non-model animals, including human cells, can be found, for example, in McCreath etal., 2000; Sedivy et al., 1999; Hanson & Sedivy, 1995; among other places. Representative protocols for gene targeting in plant cells can be found, for example, in Risseeuw etal., 1995; Kempin et al., 1997; Offringa & Hooykaas, 1995; and Hanin et al., 2001 ; among other places.

Representative methods for preparing a marked chromosome pair via gene targeting are described in Example 1. See also Figure 4. H.C. Selectable Markers

The term "selectable marker" refers to a peptide or polypeptide whose presence can be readily detected in a heterologous cell when a selective pressure is applied to the cell.

The term "selective pressure" refers to any condition that discerns cells expressing a selectable marker from cells lacking the same selectable marker. In representative embodiments of the presently claimed subject matter, a selective pressure can be applied via provision of a drug (e.g., an antibiotic, a nucleotide analog), provision of an enzymatic substrate (e.g., a chromogenic substrate), exposure to light of an appropriate excitation spectrum, and exposure to an affinity agent or column.

Examples of general types of selectable markers include, but are not limited to: (a) a nucleic acid encoding a gene product that provides resistance against, or otherwise inhibits, toxic compounds; (b) a nucleic acid encoding a gene product that is otherwise lacking in the recipient cell; (c) a nucleic acid encoding a gene product that suppresses an activity of another gene product; (d) a nucleic acid encoding a gene product that modulates an endogenous enzymatic activity; (e) a nucleic acid that can be detected using molecular methods; and (f) a nucleic acid encoding a gene product that is toxic in recipient cells.

In one embodiment of the presently claimed subject matter, a selectable marker comprises an expression-competent selectable marker, wherein a nucleic acid encoding the selectable marker is operatively fused to a promoter. The term "operatively linked", as used herein, refers to a functional combination between a promoter region and a nucleic acid molecule such that the transcription of the nucleic acid molecule is controlled and regulated by the promoter region. Techniques for operatively linking a promoter region to a nucleic acid molecule are known in the art.

Representative constitutive promoters useful for selectable marker expression in ES cells include a phosphoglycerate kinase I (PGK) promoter, a RNA polymerase II promoter, and a synthetic herpes simplex virus thymidine kinase (HSVtk) promoter (MCI) (Thomas & Capecchi, 1987; Soriano et al., 1991). Representative promoters that direct constitutive expression in plant cells include a Cauliflower Mosaic Virus (CaMV) 35S promoter (Chibbar et al., 1993), an actin promoter (McElroy ef al., 1990; McElroy et al., 1991), and a ubiquitin promoter (Norris et al., 1993; Taylor et al., 1993). An inducible promoter can also be used, for example when expression of the selectable marker induces cell toxicity or death. A representative inducible promoter includes, but is not limited to a chemically inducible promoter (e.g., a promoter regulated by the presence of a small molecule) and a heat-inducible promoter. Representative chemically-inducible promoters suitable for use in mammalian cells include a metallothionein promoter (Karin et al., 1984; Pellegrini etal., 1994), a tetracycline-responsive promoter (e.g., TET-OFF® and TET-ON® gene expression systems available from Clontech Laboratories, Inc. (Palo Alto, California, United States of America), and an ecdysone-inducible promoter (e.g., COMPLETE CONTROL® inducible mammalian expression system available from Stratagene (La Jolla, California, United States of America).

Representative inducible promoters suitable for use in plants include the chemically inducible PR-1 promoter (Lebel et al., 1998), an ethanol-inducible promoter (Caddick et al., 1998), and a glucocorticoid inducible promoter (Aoyama & Chua, 1997).

Representative heat-inducible promoters include, but are not limited to heat-responsive elements in heat shock genes (e.g., hsp20-30, hsp27, hsp40, sp60, hsp70, and hspθO). See Easton et al., 2000; Csermely et al., 1998; Ohtsuka & Hata, 2000; and references cited therein.

In another embodiment of the presently claimed subject matter, a selectable marker is targeted to a position in the genome such that expression of the selectable marker is controlled by an endogenous gene. See e.g., Wolfgang & Gossler, 2000; Hanks et al., 1995. Selectable markers employed in the methods of the presently claimed subject matter can confer dominant or recessive selectable phenotypes based on the host cell genotype. The term "dominant", as used herein to describe a selectable marker, refers to a selectable marker that it is effective in a naturally occurring cell. The term "recessive", as used herein to describe a selectable marker, refers to a marker that can be selected when expressed in cells that lack the corresponding endogenous gene, for example by spontaneous, induced, or targeted mutation. A variety of selectable markers have been described which act in either a dominant or recessive context, as noted herein below.

The term "selectable marker" encompasses positive selectable markers and negative selectable markers. Positive selectable markers and negative selectable markers are chosen as suited for a particular application. One skilled in the art can readily select an appropriate marker and selective pressure to enable selection of a desired subpopulation.

The term "positive selectable marker" refers to a marker that confers selection of cells expressing the marker in the presence of a selective pressure. The term "negative selectable marker" refers to a marker that confers selection of cells lacking the marker in the presence of a selective pressure. Some markers behave as positive selectable markers or negative selectable markers, depending on the selective pressure employed. For example, HPRT-positive cells are selected by growth in HAT (hypoxanthine / aminopterin /thymidine) medium. HPRT-negative cells are selected by growth in 6-thioguanine (6-TG). A selectable marker can also be used to positively select and negatively select cells comprising the marker when subjected to a same selective pressure. For example, cells expressing a fluorescent selectable marker (e.g., GFP, CFP, YFP, etc.) can be alternately recovered using a fluorescent- activated cell sorter (FACS) such as an EPICS® cell sorter available from Coulter Electronics, Inc. (Hialeah, Florida, United States of America).

The term "selectable marker" also encompasses a fusion protein comprising multiple selectable markers. For example, the β-geo fusion protein comprises a lacZ gene fused in-frame to a neo gene and shows both β- galactosidase activity and G418 resistance (Friedrich & Soriano, 1991). Additional selectable markers comprising fusion proteins are described in Abbate et al., 2001 ; Chen & Bradley, 2000; Oh et al., 2001. In one embodiment, the combination of markers permits selection in different settings, for example, both in vitro and in vivo. A selectable marker can be introduced at a particular locus or chromosomal position as best suited for a particular application. Exemplary positions of a marker include positions at or near the extremities of a chromosome arm. The term "distal chromosome marker" is used herein to describe a marker that is introduced at or near a distal tip of a chromosome arm. The term "centromeric chromosome marker" is used herein to describe a marker that is introduced at or near centromeric chromatin of a chromosome arm.

For preparation of a heterogeneous cellular library, wherein the heterogeneous cellular library will be used for phenotyping and mapping of dominant phenotypes, a selectable marker can comprise a positive selectable marker. Thus, a heterozygous cellular library can comprise a randomly mutagenized population of cells, wherein each of the cells comprises one or more marked chromosome comprising a dominant, positive selectable marker. In one embodiment, cells of a heterozygous cellular library comprise a different positive selectable marker on each of multiple marked chromosomes.

Representative dominant, positive selectable markers for mammalian cells include the bacterial aminoglycoside 3' phosphotransferase gene (neo), which confers resistance to the drug G418 in mammalian cells; the bacterial puromycin-N-acetyltransferase gene {puro), which confers resistance to the antibiotic puromycin; the bacterial hygromycln G phosphotransferase gene (hyg), which confers resistance to the antibiotic hygromycin; the bacterial blasticidin S deaminase gene (bsr); the bacterial xanthine-guanine phosphoribosyl transferase gene (gpt), which confers the ability to grow in the presence of mycophenolic acid; and the bacterial Sh ble gene (zeo), which confers resistance to the antibiotics phleomycin and ZEOCIN™ (Invitrogen Corp., Carlsbad, California, United States of America). In addition, the X-linked hypoxanthine-guanine phosphoribosyl transferase (hprt) gene serves as a dominant marker in male cells. Representative positive selectable markers useful in plant cells include herbicide resistance genes and antibiotic resistance genes. Selection markers used routinely in transformation include the nptll gene, which confers resistance to kanamycin and related antibiotics (Vieira & Messing, 1982); the bar gene, which confers resistance to the herbicide phosphinothricin (Vieira & Messing, 1982); the hph gene, which confers resistance to the antibiotic hygromycin (Blochlinger & Diggelmann, 1984); the dhfrgene, which confers resistance to methatrexate (Bourouis & Jarry, 1983); the EPSPS gene, which confers resistance to glyphosate (U.S. Patent Nos. 4,940,935 and 5,188,642); the mannose-6-phosphate isomerase gene, which provides the ability to metabolize mannose (U.S. Patent Nos. 5,767,378 and 5,994,629); and the doubly mutant protoporphyrinogen (PPO) gene, which confers resistance to the herbicide BUTAFENACIL™ (Syngenta Biotechnology Inc., Research Triangle Park, North Carolina, United States of America; Hanin etal., 2001). Additional representative dominant, positive selectable markers include fluorescent proteins (e.g., GFP, CFP, YFP), and enzymes that can catalyze formation of a fluorescent product. These selectable markers enable sorting of cells based on detectable fluorescence, for example using a fluorescence- activated cell sorter. A dominant, positive selectable marker can also comprise a peptide or polypeptide located on the cell surface, as described in PCT International Publication No. WO 95/06723. Cell surface marker polypeptides include membranous polypeptides such as LNGFR (low-affinity nerve growth factor receptor), CD24, and LDLR (low-density lipoprotein receptor). Also included are extracellulariy presented fragments of membranous polypeptides. In one embodiment, such fragments comprise ligand-binding or antigenic fragments to enable selection. Similarly, a peptide selectable marker can comprise a peptide (e.g., His₆) targeted to the cell surface using recombinant techniques known to one skilled in the art. In one embodiment, a peptide or polypeptide selectable marker is not endogenously expressed in the cells of a cellular library. Selection of cells comprising an extracellulariy presented peptide or polypeptide marker can be accomplished by use of a binding partner (e.g., an antibody, an endogenous ligand, a synthetic ligand, or any other binding partner) that specifically binds to the marker. See e.g., PCT International Publication No. WO/95/06723 and U.S. Patent No. 6,284,541.

For preparation of a heterozygous cellular library, wherein the library will be used to prepare a homozygous cellular library, each of the cells of the cellular library can comprise a marked chromosome pair comprising: (a) a distal selectable marker; and (b) an allelic pair of recombination cassettes, as described further herein below. Optionally, each recombination cassette ofthe pair comprises a dominant, positive selectable marker (e.g., any of those selectable markers noted herein above).

In another embodiment, each of the cells of the cellular library comprises a marked chromosome pair comprising: (a) a first distal selectable marker on a first chromosome; (b) a second distal selectable marker on a second homologous chromosome; and (c) an allelic pair of recombination cassettes.

A distal selectable marker can enable selection of homozygous chromosomes following induced mitotic recombination of a cell comprising the marked chromosome pair. Thus, a distal selectable marker comprises in one embodiment: (a) a dominant, negative selectable marker; or (b) a recessive, positive selectable marker.

In one embodiment of the presently claimed subject matter, a marked chromosome pair comprises a recessive, positive selectable marker at a distal position on a first chromosome, and a dominant, negative selectable marker at a distal position on a second homologous chromosome, thereby permitting recovery of genetic modifications induced on each chromosome of the marked chromosome pair.

Representative dominant, negative selectable markers that confer cytotoxicity include, but are not limited to gpt, which confers cytotoxicity in the presence of (6-thioxanthine) 6-TX; and HSVtk, which confers cytotoxicity in the presence of gancyclovir or 2'-fluoro-2'-deoxy-1-β-D-arabinofuranosyl-5-iodo- uracil (FIAU). Expression of the dominant, negative selectable marker encoding diphtheria toxin A fragment operates in the absence of an applied selective pressure.

A dominant, negative selection marker can also comprise a peptide or polypeptide that is presented on the cell surface in analogy to peptide markers described herein above as dominant, positive selectable markers. See U.S. Patent No. 6,284,541. Recessive, positive selectable markers are used when expressed in a mutant background devoid of the marker gene function. Representative recessive, positive selectable markers include but are not limited to thymidine kinase (tk), Herpes simplex thymidine kinase (HSVtk), carbamoyl-phosphate synthetase 2 / aspartate transcarbamylase / dihydroorotase {CAD), hprt or an hprt minigene (Reid et al., 1990). II.D. In Vitro Mutagenesis

The term "mutagenizing" is used herein to refer to a method for inducing one or more genetic modifications in cellular nucleic acid material. The terms "genetic modification" and "mutation" each refer to any alteration of DNA that to a form that is different than its naturally occurring form.

Representative gene modifications include nucleotide insertions, deletions, substitutions, and combinations thereof, and can be as small as a single base or as large as tens of thousands of bases. Thus, the term "genetic modification" encompasses inversions of a nucleotide sequence and other chromosomal rearrangements, whereby the position or orientation of DNA comprising a region of a chromosome is altered. A chromosomal rearrangement can comprise an intrachromosomal rearrangement or an interchromosomal rearrangement. In one embodiment, the mutagenizing methods employed in the presently claimed subject matter are substantially random such that a genetic modification can occur at any available nucleotide position within the nucleic acid material to be mutagenized. Stated another way, in one embodiment the mutagenizing does not show a preference or increased frequency of occurrence at particular nucleotide sequences.

The methods of the presently claimed subject matter can employ any mutagenic agent including, but not limited to ultraviolet light, X-ray radiation, gamma radiation, Λ-ethyl-N-nitrosourea (ENU), methylnitrosourea (MNU), procarbazine (PRC), triethylene melamine (TEM), acrylamide monomer (AA), chlorambucii (CHL), melphalan (MLP), cyclophosphamide (CPP), diethyl sulfate (DES), ethyl methane sulfonate (EMS), methyl methane sulfonate (MMS), 6- mercaptopurine (6-MP), mitomycin-C (MMC), /V-methyl-/V-nitro-/V- nitrosoguanidine (MNNG), ³H₂O, and urethane (UR). See e.g., Rinchik, 1991 ; Marker ef al., 1997; and Russell, 1990. Additional mutagenic agents are described at http://dir.niehs.nih.gov/dirtb/dirrtg/chemicalsstudiedindex2.htm.

In one embodiment of the presently claimed subject matter, ENU is employed as a mutagenic agent. ENU can efficiently produce point mutations and, less frequently, small deletions at sites throughout the genome. These types of genetic modifications can lead to reduced or lost gene function (including dominant negative gene function), up-regulated gene function, and altered or new gene function.

The term "mutagenizing" also encompasses a method for altering (e.g., by targeted mutation) or modulating a cell function, to thereby enhance a rate, quality, or extent of mutagenesis. For example, a cell can be altered or modulated to thereby be dysfunctional or deficient in DNA repair, mutagen metabolism, mutagen sensitivity, genomic stability, or combinations thereof.

Thus, disruption of gene functions that normally maintain genomic stability can be used to enhance mutagenesis. Representative targets of disruption include, but are not limited to DNA ligase I (Bentley et al., 2002) and casein kinase I (U.S. Patent No. 6,060,296).

The frequency of genetic modification upon exposure to one or more mutagenic agents can be modulated by varying dose and/or repetition of treatment, and can be tailored for a particular application. For example, if subsequent phenotypic screening involves identification of a rare drug resistance phenotype, then a frequency of genetic modification can be selected whereby multiple mutations are induced on each chromosome. Similarly, if the library will be used to screen for a more general differentiation phenotype, then the dose and administration of mutagen can be varied to generate a relatively fewer number of genetic modifications per chromosome. In one embodiment, the treatment dose and regimen does not induce substantial cytotoxicity.

II.EΞ. Induced Mitotic Recombination

The presently claimed subject matter further provides methods for homozygosing a heterozygous library of the presently claimed subject matter via induced mitotic recombination. In a representative embodiment of the presently claimed subject matter, the method comprises: (a) providing a heterozygous cellular library comprising a plurality of isolated parent cells; (b) inducing site-specific mitotic recombination in the plurality of isolated parent cells; (c) culturing the plurality of isolated parent cells, whereby a population of daughter cells is produced; and (d) selecting daughter cells comprising a homozygous genetic modification, whereby a homozygous cellular library is prepared.

The term "recombination" refers to a process wherein chromosomes are broken and rejoined in new combinations. The term "mitotic recombination" refers to recombination between or among homologous chromosomes during mitotic cell division.

The term "induced", as used herein to describe a type of mitotic recombination, refers to a process whereby mitotic recombination is elicited at predetermined chromosomal sites via provision of a site-specific recombinase. In one embodiment, induced mitotic recombination results in exchange of substantially the entire region of a marked chromosome arm with the allelic region of a marked homologous chromosome arm, as described further herein below. Thus, the term "inducing mitotic recombination" refers to provision of a site-specific recombinase to a cell, whereby mitotic recombination is induced. The term "chromosomal site", as used herein to describe a site at which mitotic recombination is induced, refers to a position on a chromosome characterized by a unique nucleotide sequence that is recognized by a site- specific recombinase. A chromosomal site for induced mitotic recombination can be predetermined by modifying genomic sequence at a given site to include a recombination site. Such modifications can be made using gene targeting methods, as described herein above. See also Example 1.

A chromosomal site for site-specific mitotic recombination is also referred to herein as a "recombination site." Representative recombination sites for include lox, FRT, and att sites, which mediate mitotic recombination via Cre recombinase, FLP recombinase, and Int recombinase, respectively, as described further herein below.

In one embodiment, a combination of a site-specific recombinase and recombination sites is selected such that a first recombination event is stable, thereby favoring recovery of homozygous chromosomes. A first recombination event is referred to as "stable" when it does not readily recombine again. Stated another way, the recombination sites are excision-resistant following a first recombination event.

Stability of a first recombination can be favored by: (a) recombination sites that recombine to generate a recombination site that resists excision; (b) site-specific recombinases that display compromised excision functions; (c) a presence or absence of modulators of a site-specific recombinase, for example accessory proteins of a site-specific recombinase; and (d) combinations thereof.

For example, the λ Int recombinase, when provided in the absence of the Xis accessory protein, mediates recombination at affP and attB sites that is essentially irreversible (Thorpe & Smith, 1998). As another example, Cre- induced recombination at mutant lox sites loxββ and lox71 generates a modified lox site that is unable to subsequently recombine despite the presence of Cre. Site-Specific Recombinases. The term "recombinase" generally refers to an enzyme that catalyzes recombination.

The term "site-specific", as used herein to describe a type of recombinase or a type of recombination, refers to a process of breaking and joining chromosomes at prescribed positions, which are referred to herein as "recombination sites." In accordance with the methods of the presently claimed subject matter, a site-specific recombinase mediates recombination at prescribed sites on homologous chromosomes.

Any suitable site-specific recombinase can be used to induce mitotic exchange in a cell of a cellular library, provided that the chromosomes of the cell comprise recombination sites recognized by the site-specific recombinase of choice.

Thus, a site-specific recombinase comprises in one embodiment a resolvase-type recombinase (Stark et al., 1989; Maeser & Kahmann, 1991 ; Oram ef al., 1995) that mediates intrachromosomal recombination. In another embodiment, a site-specific recombinase employed in the methods of the presently claimed subject matter comprises an integrase-type recombinase including, but not limited to prokaryotic plasmid recombinases, yeast plasmid recombinases, bacterial recombinases of the Xer and Fim families, and phage recombinases (Esposito & Scocca, 1997; Grainge & Jayaram, 1999).

Representative integrase-type recombinases include but are not limited to an Int recombinase, including those derived from phage phiC31 (Groth etal., 2000; Thyagarajan et al., 2001), phage R4 (Olivares et al., 2001), or from phage HK022 (Kolot et al., 1999); a Cre recombinase (Sauer & Henderson, 1988; Nagy, 2000), a FLP recombinase (Fiering etal., 1993; Sadowski, 1995; Seibler et al., 1998), and an integrase-type recombinase from bovine leukemia virus (Tanaka etal., 1998).

In one embodiment of the presently claimed subject matter, a site- specific recombinase used in the disclosed methods comprises a Cre recombinase. Representative Cre recombinases can comprise amino acid sequences disclosed as any one of GenBank Accession Nos. P06956, CAD22449, CAD22450, CAD22957, CAD22958, CAD22959, CAC51201 , AAL31698, and JC72 3, which are herein incorporated by reference in their entirety.

The term "site-specific recombinase" also encompasses functional variants, fragments, and fusion proteins of any of the foregoing recombinases. Thus, a recombinase used in accordance with the methods of the presently claimed subject matter can also comprise a recombinase that has been mutagenized to produce a recombinase with altered properties as desired for a particular application. For example, a site-specific recombinase can comprise enhanced FLP (FLPe), which was selected following a protein evolution strategy to improve thermolability of FLP (Buchholz ef al., 1998; Schaft et al., 2001 ). See also Sclimenti etal., 2001. A site-specific recombinase can also be modified to improve expression and inducibility, for example by optimization of codon usage as described in Koresawa et al., 2000; in European Patent 1170354; and in PCT International Publication No. WO 02/04609.

In one embodiment, a site-specific recombinase employed in the methods of the presently claimed subject matter can mediate recombination in the absence of accessory factors. However, additional factors that contribute to or modulate a recombinase activity can be co-administered with a recombinase of the presently claimed subject matter. For example, integrative recombination induced by bacteriophage λ Int recombinase involves the integration host factor (IHF) protein (Landy, 1993). To induce mitotic recombination in cells of a heterozygous cellular library of the presently claimed subject matter, a site- specific recombinase is provided to cells of the library in any manner sufficient to induce mitotic recombination. In one embodiment, the method is amenable to high-throughput formats.

In one embodiment of the presently claimed subject matter, cells are transfected with a nucleic acid molecule encoding a site-specific recombinase, whereby the site-specific recombinase is expressed and can mediate mitotic recombination. The transfected nucleic acid can be maintained as an extrachromosomal construct or can be stably integrated into the genome. Any suitable method for the introduction of a nucleic acid can be used, including transfection, microinjection, electroporation, infection with viral vectors, microparticle bombardment, etc. A representative protocol for introducing a nucleic acid molecule encoding a recombinase into a cell by electroporation is described in Liu ef al., 2002. As another example, representative protocols for infecting a cell with a viral vector encoding a site-specific recombinase are described in Kanegae et al., 1995 and in Rinaldi etal., 1999.

In one embodiment of the presently claimed subject matter, a polypeptide comprising a site-specific recombinase is provided to cells of a cellular library. For entry into cells, a site-specific recombinase comprises in one embodiment a cell-permeable, site-specific recombinase.

The term "cell permeable", as used herein to describe a site-specific DNA recombinase, refers to a recombinase that is able to transduce cell membranes. While not intended to be limited to any particular theory of operation, the translocation can occur in a receptor-independent and transporter-independent manner.

When applied exogenously to a cell, a cell-permeable recombinase can enter the cell and mediate mitotic recombination of cellular DNA. Following a temporal duration sufficient for mitotic recombination, a cell-permeable recombinase can be removed by washing the cells in culture medium lacking the recombinase. Thus, methods for chromosomal exchange employing a cell- permeable recombinase include desirable features for controlling recombinase activity in a cell.

A cell-permeable recombinase can comprise a fusion polypeptide comprising a recombinase (e.g., any one of the site-specific recombinases identified herein above) fused to a membrane translocating sequence (MTS). For example, a cell-permeable recombinase can comprise a Cre recombinase fused to the MTS of Kaposi fibroblast growth factor as described by Jo et al., 2001. See also Example 4.

The term "membrane translocation sequence" generally refers to an amino acid sequence that, in the context of a larger protein, mediates translocation of the protein across a membrane. Representative membrane translocation sequences that can be used to construct a cell-permeable recombinase include, but are not limited to MTSs of fibroblast growth factors (Lin etal., 1995; Tarantini etal., 1998; Keresztes & Boonstra, 1999), lactoferrin (He & Furmanski, 1995), VP22 (Elliott & O'Hare, 1997), ANTP (Joliot et al., 1991), TAT (Frankel & Pabo, 1988; Green & Loewenstein, 1988; Schwarze et al., 1999), engrailed (Joliot et al., 1998), and Hoxa-5 (Chatelin et al., 1996). Synthetic MTSs can also be used to construct a cell-permeable recombinase (Oehlke ef al., 1998; Pooga et al., 1998a; Pooga et al., 1998b; Scheller et al., 1999).

Representative methods and considerations for constructing a fusion protein comprising a MST can be found in, for example, Derossi ef al., 1998; Lindgren etal., 2000; Prochiantz, 2000; and the references cited herein above, among other places.

Recombination Cassettes. To provide for induced mitotic recombination, a heterozygous cellular library can comprise a marked chromosome pair comprising: (a) a first chromosome comprising a first recombination cassette; and (b) a second homologous chromosome comprising a second recombination cassette, wherein the first recombination cassette and the second recombination cassette are allelic.

The term "recombination cassette" refers to a nucleic acid molecule comprising a site for site-specific recombination, i.e. a nucleotide sequence at which a site-specific recombinase mediates strand breakage and rejoining. Representative recombination sites include a lox site, an FRT site, and an att site, as described further herein below.

The term "allelic" refers to a same chromosomal position on homologous chromosomes. For example, a first position is allelic to a second position, wherein a first position is located within a gene or intergenic region, and wherein a second position is located within the same gene or intergenic region on a homologous chromosome.

The term "lox site" refers to a nucleotide sequence that mediates mitotic recombination by a Cre recombinase, for example a naturally occurring /o site, loxP. A lox site typically comprises an about 34 base pair sequence that includes a core spacer sequence of about 8 base pairs and a pair of palindromic sequences that flank the core spacer, each palindromic region comprising about 8 base pairs to about 13 base pairs. See Hoess et al., 1982. The term "FRT site" refers to a nucleotide sequence that mediates mitotic recombination by a FLP recombinase. An FRT site is similar to a lox site, comprising a minimal sequence of about 34 base pair sequence that includes a core spacer sequence of about 8 base pairs and a pair of palindromic sequences that flank the core spacer, each palindromic region comprising about 8 base pairs to about 13 base pairs (McLeod ef al., 1986). Typically, a palindromic sequence of an FRT site includes a pyrimidine tract, and a spacer region includes a predominance of AT base pairs (Umlauf & Cox, 1988). This minimal sequence is also flanked on one side by an about 13 base pair inverted repeat, which can promote the efficiency of recombination (Jayaram, 1985). A lox site and an FRT site each comprise a directionality, which is defined by the asymmetry of the core spacer. The outcome of recombination reactions depends on the relative orientation of the two recombining sites. Conventions for describing the directionality of a lox site and an FRT site are described in Snaith et al., 1995. In accordance with a representative method of the presently claimed subject matter, a marked chromosome pair of a cell of a cellular library can comprise a pair of lox sites or a pair of FRT sites, wherein each pair of recombination sites comprises a same orientation. See Figures 3, 4, and 5B.

The term "aff site" refers to a nucleotide sequence that mediates mitotic recombination by an Int recombinase. Representative att sites include attB, attP, attL, and attR. attB is an about 25 base pair sequence containing two 9 base pair core-type Int binding sites and a 7 base pair overlap region. attP is an about 240 base pair sequence containing "core-type" Int binding sites, "arm- type" Int binding sites, and binding sites for accessory recombination proteins. See Landy, 1989. Phage Int recombinases catalyze recombination between an affSsite and an attP site. Recombination results in hybrid sites attL and attR, which are refractory to further recombination unless a excisionase protein is supplied (Thorpe & Smith, 1998). The terms "lox site", "FRT site", and

"att site" also encompass recombination sites that have been modified from their naturally occurring sequence and which mediate recombination by a Cre recombinase, a FLP recombinase, or an Int recombinase, respectively. For example, a recombination site can be modified to enhance specificity of recombination, efficiency of recombination, and stability of a recombination event. A modified recombination site can be generated using standard techniques in the field, including but not limited to chemical synthesis of a modified site and mutagenesis. See e.g., Lee & Saito, 1998; Schlake & Bode, 1994; and PCT International Publication Nos. WO 01/23545 and WO 99/25851. As noted herein above, recombination sites can be selected to favor stability of a first recombination event. Thus, in one embodiment of the presently claimed subject matter, a cell of a cellular library comprises a marked chromosome comprising a first recombination cassette and a second allelic recombination cassette, wherein the first recombination cassette comprises a lox66 site, and wherein the second recombination cassette comprises a lox71 site. Lox66 recombines with lox71 to produce a mutant lox site that will not recombine with itself or a wild type lox? site (Albert ef al., 1995; Araki et al., 1997).

In another embodiment of the presently claimed subject matter, a cell of a cellular library comprises a marked chromosome comprising a first recombination cassette and a second allelic recombination cassette, wherein the first recombination cassette comprises an attB site, and wherein the second recombination cassette comprises an attP site.

A recombination cassette used in accordance with the methods of the presently claimed subject matter can further comprise a plurality of recombination sites to thereby promote the frequency of recombination. As one example, a recombination cassette can comprise a pair of recombinase sites that flank an arbitrary intervening sequence. See e.g., Liu et al., 2002.

Similarly, a marked chromosome can further comprise multiple recombination cassettes in substantially close proximity on a chromosome to thereby promote the efficiency of recombination. For example, two or more recombination cassettes can be located within in one embodiment a region spanning about 100 kilobases (kb), in another embodiment a region spanning about 10 kb, in another embodiment a region spanning about 1kb, and in still another embodiment a region spanning about 100 base pairs. A recombination cassette can optionally comprise a dominant, positive selectable marker such that the presence of the selectable marker indicates the presence of a chromosomal site for induced mitotic recombination. A selectable marker included in a recombination cassette also facilitates selection of cells comprising a homozygous mutation following induced mitotic recombination.

Thus, a cell of a heterozygous library useful for preparing a homozygous cellular library comprises in one embodiment a marked chromosome pair comprising: (a) a first recombination cassette, wherein the first recombination cassette comprises a first recombination site and a first selectable marker proximal to the first recombination site; and (b) a second recombination cassette, wherein the second recombination site comprises a second recombination site and a second selectable marker distal to the second recombination site. The terms "proximal" and "distal" are used herein according to convention in the art to describe relative positions along a chromosome arm. The term "proximal" refers to a first position that is closer to a centromere when compared to a second position on a same chromosome arm. Conversely, the term "distal" refers to a first position that is further from a centromere, or closer to a chromosomal tip, when compared to a second position on a same chromosome arm.

To optimize the size of chromosomal regions that are exchanged during induced mitotic recombination, recombination cassettes are located in one embodiment at chromosomal positions that are substantially close to the centromere, also referred to herein as a "centromeric" position.

The phrase "substantially close", as used herein to describe to a proximity of a recombination cassette to a centromere, refers to a chromosomal distance in one embodiment less than about 50% of a chromosome arm, in another embodiment less than about 25% of a chromosome arm, in another embodiment less than about 10% of a chromosome arm, in another embodiment less than about 5% of a chromosome arm, in another embodiment less than about 1 % of a chromosome arm, and in still another embodiment less than about 0.1% of a chromosome arm. A chromosomal distance can be measured, for example in map units, as described further herein below under the heading "Gene Mapping". Stated another way, the phrase "substantially close" can refer to a position within or adjacent to a satellite DNA sequence of centromeric chromatin (e.g., an alphoid).

The frequency of induced mitotic recombination can vary according to the chromosomal location of the recombination sites. See e.g., Herault ef al., 1998 and Liu et al., 2002. Thus, a recombination cassette employed in the methods of the presently claimed subject matter can be introduced at a chromosomal site (e.g., by gene targeting) that mediates induced mitotic recombination at a frequency of in one embodiment at least about 0.1%, in another embodiment at least about 1 %, in another embodiment at least about 10%, and in yet another embodiment at least about 20%. As required, position- dependent frequency of induced mitotic recombination can be empirically determined and using standard methods in the art. M In Vitro Phenotyping The presently claimed subject matter further provides methods for screening cellular libraries prepared as disclosed herein to assess a phenotype of interest. Replica plates of a heterozygous cellular library or a homozygous cellular library can be prepared, such that a single library can be evaluated in multiple, different phenotypic assays. The in vitro phenotyping methods of the presently claimed subject matter advantageously eliminate the requirement for generation of whole organisms in which to evaluate gene function.

The term "phenotype" refers to any observable property of an organism that is dependent upon the genome of the organism. A phenotype can be further characterized as modulated by a non-genetic factor, an interaction between two or more non-genetic factors, an interaction between a genetic locus and a non-genetic factor, or an interaction between two or more genetic loci and non-genetic factors. A non-genetic factor comprises an environmental condition or exposure, for example a habitat condition, a level of activity or exercise, diet, a drug treatment, and combinations thereof. The term "phenotype", alternately expressed herein as "phenotyping", also refers to a method for assaying a phenotype. A phenotyping method is in one embodiment amenable to high-throughput formats such that a phenotype of each of the cells of the library is rapidly assessed. Cells that display a phenotype of interest can be selected, and subsequent phenotyping can be performed in chimeric or cloned organisms, as described herein below.

PCT International Publication No. WO 99/67361 to Wovchik et al. discloses a method for screening a heterozygous cellular library by detecting the occurrence of mutations in a gene of interest. For example, a gene of interest can be amplified by PCR, and the sequence of the amplified product can be analyzed to identify genetic modifications. Thus, a screen conducted according to the method of Wovchik et al. requires determination of a mutant genotype.

In contrast to Wovchik ef al., the presently claimed subject matter provides that cellular libraries of the presently claimed subject matter can be screened based on a resulting phenotype, which can be caused by genetic modification of any locus segregating on a marked chromosome. A phenotypic screening approach as disclosed herein also enables discovery of interacting loci that contribute to a phenotype (e.g., in the case of oligogenic and complex traits).

In one embodiment of the presently claimed subject matter, a screening method employs a heterozygous cellular library, wherein each of the cells ofthe cellular library comprises a marked chromosome. Phenotyping of a heterozygous cellular library is used to identify dominant mutations that confer a phenotype of interest.

In another embodiment of the presently claimed subject matter, a screening method employs a homozygous cellular library. In this case, a phenotypic screening of the library identifies recessive mutations that confer a phenotype of interest. In accordance with the presently claimed subject matter, in vitro phenotyping methods of the presently claimed subject matter are conducted in multi-well plate format. For example, a replica of a cellular library maintained in a multi-well plate can be used directly for phenotyping assays.

The cellular libraries of the presently claimed subject matter can be screened in any microplate format, including a 96-well microplate as well as higher density formats such as 384-well, 560-well, 864-well, 1 ,536-well, and 3,456-well microplates. Representative protocols and instrumentation for conducting cell-based assays in high density formats are described in, for example, Kolb & Neumann, 1997; Dias et al., 1998; Maffia et al., 1999; and Mere etal., 1999, among other places.

The disclosed serial process of generating a cellular library, phenotyping the library, and selecting one or more genetic variants that display a phenotype of interest, can be repeated as desired to uncover multiple, interacting genetic loci. For example, a mutant cell of a homozygous cellular library, which bears a modification of a first genetic locus that confers a phenotype of interest, can further be expanded and mutagenized as described herein above. The resulting derivative cellular library can then be used to perform a same phenotyping assay, whereby a second, interacting genetic locus is identified. III.A. In Vitro Differentiation

As noted herein above, a cellular library of the presently claimed subject matter can comprise precursor cells or stem cells, which are induced to differentiate prior to conducting a phenotyping assay. Differentiation of a precursor cell or a stem cell can be induced by any suitable method including, but not limited to provision of growth factors, provision of an inducing substrate, and co-culture with other cell types In one embodiment, heterogeneity among the differentiated cells is limited, such that the differentiated population is substantially a single cell type.

For example, mammalian ES cells can be induced to develop as differentiated cardiocytes (Wobus et al., 1991 ; Maltsev et al., 1993; Miller- Hance etal., 1993; Maltsev etal., 1994; Wobus etal., 1997), myocytes (Miller- Hance etal., 1993; Rohwedel etal., 1994; Rose etal., 1994), neural cells (Bain et al., 1995; Fraichard ef al., 1995; Strubing etal., 1995; Okabe et al., 1996), hematopoietic cells (Wiles & Keller, 1991 ; Hole & Smith, 1994; Keller, 1995), adipocytes (Dani ef al., 1997), epithelial cells (Bagutti ef al., 1996), endothelial cells (Risau etal., 1988), and vascular smooth muscle cells (Risau etal., 1988; Weitzer etal., 1995; Drab etal., 1997).

Methods for inducing cellular differentiation in vitro are known in the art.

Representative protocols can be found in the references to particular cell types cited herein above. See also U.S. Patent Nos. 6,322,784; 6,294,346;

6,033,906; 6,093,531 ; 6,129,911 ; 6,117,675; and 6,001 ,654; and PCT

International Publication Nos. WO 01/29206, WO 00/28000, and WO 00/27995.

III.B. Phenotypes

Any detectable or measurable quality can be used to phenotype a cellular library of the presently claimed subject matter. Representative phenotypes that can be assessed in cultured cells include but are not limited to a visible phenotype (e.g., a morphological phenotype), a viability phenotype, a molecular phenotype, a differentiation phenotype, a cell behavioral phenotype, a susceptibility phenotype, a resistance phenotype, and combinations thereof, as described further herein below. These descriptive categories are not strictly defined and are also not mutually exclusive. Representative examples described herein below are exemplary and are not intended to limit the type of assay that can be performed. Cell Viability Phenotype. Any clone that is not recovered following induced mitotic recombination and selection comprises a candidate cell lethal mutation (Figure 5A). Cell lethality can also be assayed in response to an environmental condition.

A cell lethal phenotype can be detected by observing a reduced number or absence of viable cells. Viability can be assessed, for example, by providing a substrate for mitochondrial dehydrogenase, wherein metabolism of the substrate by living cells can be detected colorimetrically. Other representative viability indicators include 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), 2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5- carboxanilide (XTT) (available from Sigma, St. Louis, Missouri, United States of America), and methylene blue. MTT and XTT are metabolized only in living cells to produce blue and orange formazan products, respectively. Methylene blue is decolorized by dehydrogenase activity of living cells. See U.S. Patent No. 5,858,687. Molecular Phenotypes. The term "molecular phenotype" refers to a detectable feature of molecules in a cell or organism. Representative molecular phenotypes include, but are not limited to a level of gene expression (e.g., a level of RNA or a level of protein), a protein modification, a protein activity (e.g., an enzyme activity), a level of lipid, production of a lipid type, a lipid modification, a level of carbohydrate, production of a carbohydrate type, a carbohydrate modification, and combinations thereof.

Methods for observing, detecting, and quantifying molecular phenotypes comprising altered levels of mRNA transcripts are well known to one skilled in the art. SeeAusubel, 1995; Innis, 1990; Koduri & Poola, 2001. Immunochemical approaches can be used to characterize a protein level or a protein structure. For example, techniques for detecting antibody-antigen conjugates or complexes include but are not limited to centrifugation, affinity chromatography and other immunochemical methods. See e.g., Manson, 1992; Ishikawa, 1999; Law, 1996; Chan, 1996; Liddell & Weeks, 1995; Masseyeff et al., 1993; Walker & Rapley, 1993; and references cited therein. Additional methods for molecular analysis that can be used in a phenotyping assay of the presently claimed subject matter include high performance liquid chromatography (HPLC), capillary electrophoresis, and mass spectrometry, which can be adapted for high-throughput analyses.

The term "mass spectrometry" as used herein refers to techniques including but not limited to gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), laser-desorption mass spectrometry (LD-MS), matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), time-of-flight mass spectrometry (TOF-MS), electrospray ionization mass spectrometry (ESI-MS); tandem mass spectroscopy, field release mass spectrometry, and combinations thereof. See e.g., Maurer, 2000; Karas et al., 2000; Kowalski & Stoerker, 2000; Griffiths ef al., 2001 ; U.S. Patent Nos. 6,107,623; 6,104,028; 6,093,300; 6,057,543; 6,017,693; 6,002,127; 5,118,937; 5,952,654; and references cited therein.

Determination of a molecular phenotype can also comprise a reagent- based assay, where a signal, for example a spectroscopic signal or a fluorescent signal, is measured in response to provision of a chromogenic or fluorogenic substrate. For example, chromogenic and fluorogenic substrates can be used to assay a variety of enzyme activities, including alcohol dehydrogenases, aldolases, lipases, amidases, epoxide hydrolases and phosphatases. See e.g., Wahler & Reymond, 2001 , and references cited therein.

Common research equipment has been developed to perform high- throughput detection of spectroscopic and fluorescent signals, including instruments from GSI Lumonics (Watertown, Massachusetts, United States of America), Amersham Pharmacia Biotech/Molecular Dynamics (Sunnyvale, California, United States of America), Applied Precision Inc. (Issauah, Washington, United States of America), and Genomic Solutions Inc. (Ann Arbor, Michigan, United States of America).

Differentiation Phenotypes. The term "differentiation phenotype" generally refers to the ability of a precursor cell or a stem cell to generate one or more differentiated cell types. Particular cell types can be assessed, for example, by morphological inspection and/or determination of a molecular phenotype as described herein above.

Cell Behavioral Phenotypes. The term "cell behavioral phenotype" generally refers to cellular activities such as cell adhesion or attachment to a surface, cell aggregation, cell motility, cell division, cell growth, cell tropism or aversion, etc.

For example, a cell behavioral phenotype can comprise a frequency of beating activity in cardiomyocytes as described by Wobus ef al., 2001.

A cell behavioral phenotype comprising a rate of cell growth can be measured, for example, by quantifying a cellular marker (e.g., an amount of antigen). See U.S. Patent Nos. 5,912, 132 and 5,707,798.

Cell division can be measured by quantification of bromo-deoxyuridine incorporation or by Hoechst-ethidium bromide staining as described by Pollard & Walker, 1997.

Representative assays for measuring chemotaxis, for example chemotaxis to chemokines, are described in Proudfoot ef al., 2000.

Physiological Phenotypes. The term "physiological phenotype" generally refers to the processes of ion conductance across a cell membrane.

Representative measures of a physiological phenotype include, for example, a resting membrane potential, an amplitude or frequency of evoked potentials, ion permeability across a membrane, etc.

A physiological phenotype can be assayed using any technique known in the art, including extracellular single unit voltage recording, intracellular voltage recording, voltage clamping, and patch clamping. Representative methods for physiological analysis can be found, for example, in Sakmann & Neher, 1995; DeFelice, 1997; and U.S. Patent No. 6,174,690.

A physiological phenotype can also be assayed by determining the flux of labeled tracer ions, for example as described by Catterall ef al., 1981 and by Reith, 1990.

A variety of fluorescent molecules have been described that respond to changes in intracellular calcium concentration, membrane potential, and pH, and can also be used as indicators of receptor stimulation and ion channel activation. See e.g., Gonzales etal., 1999; Denyer etal., 1998; and references cited therein. Specialized Cell Function Phenotypes. For cells with specialized functions, a phenotyping assay can also comprise a determination of a cellular activity. As one example, a phenotyping assay can be employed to determine a level of T cell activation or cytotoxicity as described by Kearse, 2000.

Susceptibility Phenotypes. The term "susceptibility phenotype" refers to an increased capacity or risk for displaying a phenotype, including but not limited to any phenotype noted herein above. For example, a susceptibility phenotype can comprise an increased frequency of cytotoxicity in the presence of a drug.

Resistance Phenotypes. Conversely, the term "resistance phenotype" refers to an increased capacity to resist expression of a phenotype including, but not limited to any phenotype noted herein above. For example, a resistance phenotype can comprise plant viability in the presence of an herbicide. As another example, a resistance phenotype can comprise reduced susceptibility to viral infection as described by Bedard et al., 1999. jΛ Mapping of Mutant Loci

The presently claimed subject matter further provides an in vitro method for mapping a genetic modification conferring a phenotype of interest. The method comprises: (a) culturing an isolated cell comprising one or more heterozygous genetic modifications (i.e., a random genetic variant), whereby a population of recombinant cells is produced; and (b) mapping the genomes of individuals within the population of recombinant cells that display a phenotype, whereby a genetic locus that modulates the phenotype is identified.

The term "recombinant cell" is used herein to refer to a cell that has undergone a random (non-induced) mitotic recombination event to make a chromosome that comprises heterozygous segments and homozygous segments.

Optionally, the method can further comprise selecting from among the population of recombinant genetic variants a subpopulation comprising a selectable marker, to thereby select cells comprising the genetic modification of interest.

As described herein above, a cell comprising a random genetic variant can be generated via random mutagenesis and typically comprises a heterozygous genetic modification. Thus, expansion of a cell derived from a heterozygous cellular library will produce a population of different cells as a result of mitotic recombination.

In one embodiment of the presently claimed subject matter, the culturing further comprises contacting the culture with an agent to promote mitotic recombination during expansion of the culture. Representative agents include inhibitors of DNA repair, such as a helicase inhibitor. For example, inhibitors of RecQ helicases, including porphyrin derivatives such as meso-tetra (/V-methyl- 4-pyridyl) porphine tetra tosylate (T4) and Λ/-methyl mesoporphyrin IX (NMM), can be used to promote mitotic recombination as described in Example 6.

The terms "mapping" and "gene mapping" are used interchangeably to refer to progressive resolution of genomic sequence conferring a phenotype. A typical mapping experiment employs linkage analysis of a target locus and genetic polymorphisms. The results of a mapping method can be expressed as map units or centimorgans.

The term "polymorphism" refers to the occurrence of two or more genetically determined alternative sequences or alleles in a population. An allelic difference can be as small as one base pair.

The terms "Morgan" and "map unit" each refer to a unit for expressing a relative distance between genes on a chromosome. One Morgan unit (M) indicates a recombination frequency of 100%. A centimorgan (cM) indicates a recombination frequency of 1 %. The term "recombination frequency" refers to a value calculated as a number of recombinants divided by the total number of progeny.

A mapping method employed in the methods of the presently claimed subject matter is selected to have sufficient power for resolution of a target locus. The term "power" as used herein refers to the probability of detecting or mapping a genetic locus. Power is in one embodiment 80%, in another embodiment 90%, in another embodiment 95%, and in still another embodiment 99%. The power of detection is correlated with target gene strength, and is optimal when genetic noise and environmental noise in the mapping population is low. Conversely, the power of detection is diminished by genetic noise and environmental noise.

The term "target gene" in the context of gene mapping refers to a gene that, when mutated, confers a phenotype of interest. The term "strength" and "target gene strength" each refer to the percent contribution of a single gene to a phenotype. Gene strength correlates with ease of genetically detecting a target gene. Relatively strong target genes are easily detected. Genes with relatively weak effects contribute to complex traits, and are often masked by environmental noise. The terms "genetic noise" and "genetic background" and "residual genotype" as used herein each refer to a level of genetic variation. In a gene mapping experiment, genetic noise is inversely correlated with genetic diversity. A level of genetic noise can be described by the equation: genetic noise =∑b_t x,- , wherein b represents gene strength or allele substitution effect, x represents genotype, and /^'represents a number of non-target genes. Thus, genetic noise represents a sum of allele substitution effects at all non-target loci contributing to a phenotype. Optimally, the genetic noise should approach zero for maximum sensitivity of gene mapping. The terms "environmental noise" and "environmental background" as used herein each refer to a level of environmental variation. In a gene mapping experiment, environmental noise is inversely correlated with experimental replication of identical genotypes. For example, environmental noise is significant when all individuals are unique. Optimally, environmental noise should approach zero for maximum sensitivity of gene mapping.

In accordance with the methods of the presently claimed subject matter, a mapping method can comprise an initial genome-wide scan using a population derived by expanding a cell comprising a random genetic variant. The progeny are genotyped to define an approximately 20 cM interval in which the target locus resides. A map location is then estimated using interval mapping or variations thereof, wherein linkage analysis is performed using additional genetic polymorphisms within the initially defined interval. Further evaluation of candidate genes within a small chromosomal interval is variably difficult depending on the resolution of the mapping and the power to detect genetic loci with small effects.

Techniques for gene mapping are well known to one skilled in the art, including linkage analysis (e.g., Wells & Brown, 2000), linkage disequilibrium analysis (Kruglyak, 1999), restriction landmark genomic scanning (RLGS) (Akiyoshi ef al., 2000), and radiation hybrid mapping (Schuler et al., 1996; Van Etten etal., 1999). Any suitable mapping technique can be used, and it will be appreciated by one of skill in the art that no particular choice is essential to or a limitation of the presently claimed subject matter.

An exemplary method for gene mapping is linkage analysis whereby a phenotype is correlated with one or more detectable polymorphisms including but not limited to restriction fragment length polymorphisms (RFLPs) (Lander & Botstein, 1989), short tandem repeat polymorphisms (STRPs), short sequence length polymorphisms (SSLPs) (Dietrich et al., 1996), microsatellite markers (Schalkwyk et al., 1999), and single nucleotide polymorphisms (SNPs) (Brookes, 1999).

An exemplary technique for linkage analysis is detection of SNPs. The density of SNP markers in a mammalian genome is estimated to be about 1 SNP per 1 kb of sequence. See Collins etal., 1998. Several approaches can be used for typing SNPs, including homogenous hybridization assays (Livak et al., 1995), oligonucleotide ligation assays (Chen et al., 1998), matrix-assisted laser desorption time-of-flight mass spectrometry (MALDl-TOF) (Kwok, 1998; Ross etal., 1998), high performance liquid chromatography (HPLC) (Schriml ef a/., 2000), fluorescence polarization (Chen et al., 1999), array-based technologies (Cronin et al., 1996; Hacia ef al., 1996; Pastinen et al., 1997; Gentalen & Chee, 1999; Sapolsky et al., 1999), pyrophosphate minisequencing (Nyren etal., 1993), and invader methods (Griffin etal., 1999; Lyamichev etal., 1999). See also Landegren et al., 1998.

Exemplary methods for SNP detection are array-based oligonucleotide hybridization and minisequencing, described further herein below, as these techniques are amenable to high-throughput and multiplex formats. Oligonucleotide microarrays or chips can be manufactured by photolithographic synthesis of oligonucleotides onto glass slides using, for example, the AFFYMETRIX® system (Affymax Corporation of Greenford Middlesex, Great Britain) See Fodor et al., 1991 and U.S. Patent No. 5,445,934. Alternatively, oligonucleotide microarrays can be produced by gridding oligonucleotides robotically onto the surface of a slide or other solid support (Schena et al., 1996), or by using an inkjet type technology to deliver oligonucleotides to a solid support (U.S. Patent No. 5,965,352). By either method, a particular SNP is determined by a position of an oligonucleotide having an SNP in the array. To detect a SNP using a hybridization assay, genomic fragments of a test genome are amplified by PCR and labeled such that the fragment is detectable. A SNP of the test genome is determined by the formation of a detectable heteroduplex structure at an identified position in the array. To perform minisequencing reactions on chips, genomic fragments of a test genome are amplified using PCR and hybridized to an oligonucleotide microarray. Primer extension reactions including labeled nucleotides are performed on the hybridized oligonucleotide array. A SNP of the test genome is identified as a successful primer extension reaction assayed by detecting the labeled nucleotides. Alternatively, the SNP can be detected by amplification on the solid support without prior PCR.

To confirm mapping analyses, genomic clones within the mapped region, for example a BAC clone, can be transfected into mutagenized cells to thereby recover a wild type phenotype. Regional cloning based on the genetic map position can be used to clone genes residing at the locus using methods known in the art. Alternatively, an integrated gene and physical map framework can be used to reference one or more genes at the mapping position, which can then be cloned using standard methods. See Klysik etal., 1999. V. Assays of Gene Function in Whole Organisms

In one embodiment of the presently claimed subject matter, a cellular library comprises a plurality of ES cells, and a subset of ES cells are selected for further study following determination of an in vitro phenotype. To evaluate an organismal phenotype, a selected cell can be used to produce a genetic mosaic, a chimera, a cloned non-human organism, or combinations thereof. A plant ceil that can be induced to form callus can be similarly used to generate plants for phenotypic analysis.

The term "genetic mosaic" refers to an organism comprising genetically related cells of more than one genotype. For example, a genetic mosaic organism can be generated by transplantation of mutagenized cells, or mutagenized and homozygosed cells, into a host organism from which they were originally derived.

The term "chimera" refers to an organism comprising cells from different sources. A genetic mosaic organism can be produced using the regenerative methods described herein below.

The term "clone" is used herein to describe a regenerated organism, wherein all the cells of the organisms are genetically identical. Non-human animals can be cloned by nuclear transfer, as described herein below. Plants are readily regenerated using callus cells, also described herein below.

In one embodiment of the presently claimed subject matter, a chimeric animal can be generated by injection of ES cells into intact blastocysts or morula as described by Bradley etal., 1984. Representative protocols, markers for chimerism, and breeding strategies can be found in, for example, Papaionannou & Johnson, 2000.

A chimeric animal can also be generated by using ES cell aggregates as described by Nagy ef al., 1990. Additional methods can be found in, for example, Bradley, 1987 and Nagy & Rossant, 2000.

In another embodiment of the presently claimed subject matter, cells of a cellular library, including stem cells, precursor cells, and differentiated cells, can be used to clone an animal via somatic nuclear transfer (Wilmut ef al., 1997;

Ashworth et al., 1998; Signer et al., 1998). Animals have been cloned from diverse differentiated cell types, including cells derived from mammary gland, cumulus, oviduct, granulosa, muscle, leukocyte, ear, skin, tail, and sertoli cells. Representative methods for somatic nuclear transfer can be found in, for example, Kato et al., 1998; Wells et al., 1999; Shiga et al., 1999;

Zakhartchenko etal., 1999; Hill etal., 2000; Kubota etal., 2000; Wakayama et al., 1998; Wakayama & Yanagimachi, 1999; Ogura ef al., 2000; Polejaeva et al., 2000. Methods for plant regeneration are also known in the art and can be found, for example, in U.S. Patent Nos. 6,091 ,004; 6,031 ,153; 5,986,082; and

5,792,904.

Mosaic, chimeric, and non-human cloned organisms can be studied to elucidate pathological phenotypes that are not readily detected at a cellular level including, but not limited to physiological, neurological, and organismal behavioral phenotypes. Any suitable method can be used to investigate phenotypes of mosaic, chimeric, and non-human cloned organisms, including in vivo as well as post-mortem analyses. Representative techniques include external observation, magnetic resonance imaging (MRI), computerized tomography (CT), microscopy, and methods, histological methods, enzymatic assays, biochemical assays, assays to detect changes in gene transcription, including transcription profiling of multiple genes (e.g., chip analysis). In addition, in vitro phenotypic assays described herein above can be performed using cells derived from a mosaic, chimeric, or non-human cloned organism.

General approaches for pathological analysis are described in Agrios, 1997; Porth & Kunert, 2002, and references cited therein. Specific strategies and resources for analyzing mutant mice can be found in Branson, 2001 , and references cited therein.

Examples

The following Examples have been included to illustrate modes of the presently claimed subject matter. Certain aspects of the following Examples are described in terms of techniques and procedures found or contemplated by the present co-inventors to work well in the practice of the presently claimed subject matter. These Examples illustrate standard laboratory practices of the co-inventors. In light of the present disclosure and the general level of skill in the art, those of skill will appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently claimed subject matter.

Example 1 Preparation of ES Cells Comprising Marked Chromosomes Non-recombinant inbred mouse strains C57BIJ6J and 129S8/SvEv@ J- Gpil^cHprl^b'm2 are crossed. ES cells are isolated from the resulting F1 progeny and maintained in an undifferentiated state by culturing them on a feeder cell layer.

Gene targeting methods were used to prepare ES cells comprising a pair of allelic recombination cassettes and a distal chromosome marker. A targeting vector p[Puro^r lox71] was prepared comprising a [Puro^r lox71] flanked by genomic sequences at the C57BIJ6J D4MK149 locus. A targeting vector p[lox66 β-geo] was prepared comprising a [lox66 β-geo] recombination cassette flanked by genomic sequences at the 129S8/SvEv@ J-Gp/^cHprt^b"m2D4Mit149 locus. A targeting vector comprising a distal chromosome marker was prepared using an HPRTcD A flanked by genomic sequence at the C57BL76J D4Mit51 locus. The targeting vectors were sequentially electroporated into ES cells essentially as described by Stevens, 1983. Recombinants were selected by growth in media containing puromycin, neomycin, and hypoxanthine- aminopterin-thymidine (HAT).

Example 2 Preparation of a Heterozygous Cellular Library ES cells comprising a marked chromosome pair are prepared as described in Example 1. Cells are grown in MEM culture medium supplemented with 15% heat-inactivated fetal calf serum, 1000 units/ml leukemia inhibitory factor (LIF), and 10 μM β-mercaptoethanol. Cells are grown on 100-mm petri dishes and cultured at 37°C in a humidified atmosphere of 5% CO₂ in air. For subculturing, cells are dissociated in Hank's Balanced Salt Solution (HBSS) containing 0.25% trypsin and 0.02% ethylene diamine tetraacetic acid (EDTA). Following a 3-minute incubation at room temperature, dissociated cells are resuspended in culture medium, and the number of cells is determined using a hemocytometer. Culture media and supplements are available from Invitrogen Corp., Carlsbad, California, United States of America.

Plating efficiency is determined by determining the percentage of viable cells in mature cultures. Briefly, cells are plated at a density of about 2 X 10³ cells per 100-mm petri dish, and the cells are cultured for about 6 days. The cells are then fixed in methanol and stained with 0.01% crystal violet. Viable cells are counted. Plating efficiency is expressed as a percentage of inoculated cells that are viable following culture. Culture conditions are modified as necessary to optimize plating efficiency.

Actively growing ES cells are trypsinized and plated at a density of about

5 X 10⁵ cells per T25 flask. The cells are pre-incubated for 1 day and then treated with 0.3 mg/ml to 0.5 mg/ml ENU for about 5 hours. ENU stock solutions are prepared by dissolving ENU in medium without fetal calf serum. Vigorous shaking can be used to promote dissolution. ENU stock solutions are sterilized by passing through a 0.2 μm cellulose acetate filter immediately prior to use.

Surviving ES cells are subcultured at 3:1 onto 60-mm petri dishes in the presence of primary mouse embryonic fibroblasts (PMEFs). ES cells are cultured on PMEFs for about 2 days, and then subcultured at 3:1 onto 60-mm petri dishes without PMEFs. ES cells so-prepared are then cultured for about 5 days, trypsinized, and plated at a density of about 4 X 10⁵ cells per 100-mm plate. The cells are cultured in the absence of PMEFs for about 6 days, at which time cells are individually plated in wells of a 96-well plate. The resulting plates of individual mutagenized ES cells comprise a heterozygous cellular library.

The library is cultured for a time sufficient to expand the library for the preparation of replica libraries. The replica libraries are alternately used for preparation of a homozygous cellular library (Example 4), for phenotypic screening (Example 5), and for gene mapping (Example 6). Replica libraries are cryopreserved as described in Example 3.

Example 3 Storage of Cellular Libraries Cellular libraries and replica cellular libraries are preserved by storage in a cryopreservation medium at or below -70°C. Cryopreservation media generally consists of a base medium, a cryopreservative, and a protein source. The cryopreservative and protein protect the cells from the stress of the f reeze- thaw process. For serum-containing medium, a typical cryopreservation medium is prepared as complete medium containing 10% glycerol; complete medium containing 10% DMSO (dimethylsulfoxide), or 50% cell-conditioned medium with 50% fresh medium with 10% glycerol or 10 % DMSO. For serum- free medium, typical cryopreservation formulations include 50% cell-conditioned serum free medium with 50% fresh serum-free medium containing 7.5% DMSO; or fresh serum-free medium containing 7.5% DMSO and 10% cell culture grade

DMSO. A cell suspension typically comprises about 10⁶ to about 10⁷ cells per ml is mixed with cryopreservation medium. Cellular libraries comprising ES cells can be frozen at a cellular density of about 5 X 10⁶ cells/ml of freezing medium to about 10 X 10⁶ cells/ml of freezing medium. Cells can be cooled from room temperature to a storage temperature at a rate of about -1 °C per minute. The cooling rate is controlled, for example, by placing vials containing cells in an insulated water-filled reservoir having about 1 liter liquid capacity, and placing such cube in a -70°C mechanical freezer. Alternatively, the rate of cell cooling is controlled at about -1°C per minute by submersing vials in a volume of liquid refrigerant such as an aliphatic alcohol, the volume of liquid refrigerant being more than fifteen times the total volume of cell culture to be frozen, and placing the submersed culture vials in a conventional freezer at a temperature below about -70°C. Commercial devices for freezing cells are also available, for example, the Planer Mini-Freezer R202/200R (Planer Products Ltd., Great Britain) and the BF-5 Biological Freezer (Union Carbide Corporation, Danbury, Connecticut, United States of America). In one embodiment, frozen cells are stored at or below about -70°C to about -80°C, and in another embodiment at or below about -130°C. To obtain the best possible survival of the cells, thawing of the cells must be performed as quickly as possible. Once a vial or other reservoir containing frozen cells is removed from storage, it should be placed directly into a 37°C water bath and gently shaken until it is completely thawed. If cells are particularly sensitive to cryopreservatives, the cells are centrifuged to remove cryopreservative prior to further growth.

Example 4 Preparation of a Homozygous Cellular Library A cell-permeable Cre recombinase was prepared essentially as described by Jo et al., 2001. Briefly, a recombinant nucleic acid encoding a His₆-NLS-Cre-MTS polypeptide was expressed in E. coli, and the recombinant Hisβ-NLS-Cre-MTS polypeptide was purified by affinity chromatography, as directed by the supplier of the affinity matrix (Qiagen, Valencia, California, United States of America).

A cellular library of random genetic variants, or a replica thereof, is prepared as described in Example 2. The cellular library is cultured in serum- free medium containing about 10 μM His₆-NLS-Cre-MTS for about 2 hours. The cellular library is washed in serum-free medium for about 3 hours. The cellular library is then cultured in medium containing 10 μM 6-TG. Individual cells are re-plated such that the library format is maintained, and the cells are again cultured in medium containing 10 μM 6-TG. The resulting library comprises homozygous genetic variants. An identifiable position in the library

(e.g., plate number and well number) of each random genetic variant and a homozygous genetic variant derived therefrom is preserved.

The cellular library of homozygous genetic variants is cultured for a time sufficient for the preparation of replica libraries. Replica libraries are prepared, which can then be employed in a variety of phenotypic screens (e.g., Example

5). Replica libraries cryopreserved as described in Example 3. Example 5

Phenotypic Screening

Phenotypic screening is used to select cells comprising mutations that confer a phenotype of interest. A heterozygous cellular library, prepared as described in Example 2, is screened to identify dominant mutations. A homozygous cellular library, prepared as described in Example 3, is screened to identify recessive mutations.

Example 6

In Vitro Gene Mapping

A homozygous cellular library, prepared as described in Example 4, is screened to identify mutations conferring a phenotype of interest, as described in Example 5. Cells conferring the phenotype are identified according to a position in the library (e.g., plate number and well number). The corresponding cells from the heterozygous cellular library are identified and selected for mapping. Each of the selected cells is cultured in medium containing meso-tetra

(/V-methyl-4-pyridyl) porphine tetra tosylate (T4) and Λ/-methyl mesoporphyrin

IX (NMM) (available from Porphyrin Products, Logan, Utah, United States of

America). Conditions including porphyrin concentration and the duration of culture are empirically determined so as to optimize mitotic recombination, the number of recombinants recovered, and cellular viability. Cells of the expanded culture are washed in medium and then cultured in the presence of 10 μM 6-

TG. Resistant cells are individually plated in 96-well plates, to thereby prepare a mapping population. The mapping population is used for mapping using recombination breakpoint delineation. In one embodiment, the mapping can localize the breakpoint to a 1 cM interval, and in another embodiment a 0.1 cM interval.

Once an interval has been defined, candidate genes within the region are tested for complementation. For example, a BAC clone comprising a candidate gene is introduced into a cell displaying a phenotype of interest, and rescue of the phenotype is scored. Alternatively or in addition, candidate genes are sequenced to identify genetic modifications.

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It will be understood that various details of the presently claimed subject matter can be changed without departing from the scope of the presently claimed subject matter. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation~the presently claimed subject matter being defined by the claims.