EP0264166B1

Transgenic animals secreting desired proteins into milk

Abstract

This record has no abstract on file.

EP0264166B1, drawing sheet 1 — Sheet 1 of 5

Term

Term ended

Expired 9 April 2007, 19.5 years ago.

10 claims: 2 independent, 8 dependent

1
A DNA sequence containing a gene encoding a protein, said gene being under the transcriptional control in said DNA sequence of a mammalian casein or milk serum protein promoter which does not naturally control the transcription of said gene, said DNA sequence further comprising DNA enabling secretion of said protein.
2
The DNA sequence of Claim 1, wherein said secretionenabling DNA comprises a secretion signal-encoding sequence interposed between said gene and said promoter.
3
The DNA sequence of Claim 1 wherein said milk serum protein is a whey acid protein.
4
The DNA sequence of Claim 2 wherein said signal encoding sequence is the signal encoding sequence naturally associated with said gene encoding said protein.
5
The DNA sequence of Claim 2 wherein said signal encoding sequence is the signal encoding sequence naturally associated with said mammalian casein or milk serum protein promoter.
6
The DNA sequence of Claim 1 wherein said DNA sequence includes a transcriptional stop sequence.
7
The DNA sequence of Claim 6 wherein said stop sequence is derived from SV40 virus DNA.
8
The DNA sequence of Claim 6 wherein said stop sequence is contained in the polyadenylation sequence of SV40.
9
The DNA sequence of Claim 1 wherein said protein is human tissue plasminogen activator or hepatitis B surface antigen.
10
A method for producing a protein comprising the steps of:(a) inserting into a non-human mammalian embryo a DNA sequence comprising a gene encoding said protein, said gene being under the transcriptional control of a casein or milk serum protein promoter which does not naturally control the transcription of said gene, said DNA sequence comprising DNA enabling secretion of said protein, (b) allowing said embryo to develop into an adult mammal, (c) inducing lactation in said mammal, or in a female descendant of said mammal in which said gene, promoter, and signal sequence are present in the mammary tissue genome, (d) collecting milk of said lactating mammal, and (e) isolating said protein from said collected milk.

51 paragraphs in 2 sections, as filed

0001This invention relates to transgenic animals.

0002It is possible to insert foreign genes into vertebrate embryos, and for these genes to be incorporated into the genome of the resulting animal. Insertion of the foreign genes has been carried out mechanically (by microinjection), and with the aid of retrovirus vectors (for example, as is described in Huszar et al. (1985) P.N.A.S. U.S.A 82, 8587). The animals resulting from this process are termed "transgenic." The foreign genes can be sexually transmitted through subsequent generations and are frequently expressed in the animal. In-some instances the proteins encoded by the foreign genes are expressed in specific tissues. For example, the metallothionein promoter has been used to direct the expression of the rat growth hormone gene in the liver tissue of transgenic mice (Palmiter et al., 1982 Nature 300:611). Another example is the elastase promoter, which has been shown to direct the expression of foreign genes in the pancreas (Ornitz et al., 1985 Nature 313:600). Developmental control of gene expression has also been achieved in transgenic animals, i.e., the foreign gene is transcribed only during a certain time period, and only in a particular tissue. For example, Magram et al. (1985, Nature 315:338) demonstrated developmental control of genes under the direction of a globin promoter; and Krumlauf et al. (1985, Mol. Cell. Biol. 5:1639) demonstrated similar results using the alpha-feto protein minigene.

0003In a first aspect thereof, the invention features a DNA sequence containing a gene encoding a protein, the gene being under the transcriptional control of a mammalian casein or milk serum protein promoter which does not naturally control the transcription of the gene, the DNA sequence further including DNA enabling secretion of the protein; e.g., a secretion signal-encoding sequence interposed between the gene and promoter. The promoter can be that of a milk serum protein or a casein protein, although milk serum proteins are preferred, as will be discussed in more detail below. (As used herein, "gene" refers to both genomic DNA sequences and cDNA sequences.)

0004The invention permits the production of any desired protein in an easily maintained stable, portable culture system, i.e., a living domesticated mammal, which is capable not only of producing the desired protein, but preferably of passing on the ability to do so to its female offspring as well. Secretion of the protein into the host mammal's milk facilitates purification and obviates removal of blood products and culture media additives, some of which can be toxic or carcinogenic. More importantly, protein yields will be high and production will be more cost effective and efficient.

0005The invention provides, in a third and further alternative aspect thereof, a method for producing a protein comprising the steps of: <ul id="ul0001" list-style="none" compact="compact"><li>(a) inserting into a non-human mammalian embryo a DNA sequence comprising a gene encoding said protein, said gene being under the transcriptional control of casein or milk serum protein promoter which does not naturally control the transcription of said gene, said DNA sequence comprising DNA enabling secretion of said protein,</li><li>(b) allowing said embryo to develop into an adult mammal,</li><li>(c) inducing lactation in said mammal, or in a female descendant of said mammal in which said gene, promoter, and signal sequence are present in the mammary tissue genome,</li><li>(d) collecting milk of said lactating mammal, and</li><li>(e) isolating said protein from said collected milk.</li></ul>

0006The invention is hereinafter more particularly described by way of example only with reference to the accompanying drawings, in which:- <ul id="ul0002" list-style="none" compact="compact"><li>Fig. 1 is a diagrammatic representation of the construction of an embodiment of intermediate vector in accordance with the present invention, namely pt-PA VPl-LP(K);</li><li>Fig. 2 is a diagrammatic representation of the construction of another embodiment of intermediate vector in accordance with the invention, namely pWAP (H3);</li><li>Fig. 3 is a diagrammatic representation of the construction of another vector in accordance with the invention, namely pWAP-t-PA(S);</li><li>Fig. 4 is a diagrammatic representation of the construction of another intermediate vector in accordance with the invention, namely pHbsSVA; and</li><li>Fig. 5 is a diagrammatic representation of the construction of another vector according to the invention, namely pWAP-Hbs (S).</li></ul>

DNA

S

equence

Elements

Promoter

0007The milk protein promoter can be derived from any mammalian species, and can be any promoter naturally associated with any protein which is normally secreted into mammalian milk. Generally, milk proteins are classified as the caseins, which are defined herein as the milk proteins which are present in milk in the form of micelles, and which are removed from skim milk by clotting with rennet; and the milk serum proteins, which are defined herein as the non-casein milk proteins. Whey proteins constitute the predominant fraction of the milk serum proteins, and in rodents include the protein known as whey acid protein. Whey acid protein ("WAP") is named based on its acidic isoelectric point (Piletz (1981) J. Biol. Chem. 256: 11509). Another example of a milk serum protein described in the literature is α-lactalbumin (described, along with mouse WAP, in Hennighausen and Sippel (1982) Eur. J. Biochem. 125, 131). Milk proteins are discussed in detail in Walstra and Jenness DairyChemistryandPhysics (John Wiley & Sons 1984).

0008Generally, milk serum protein promoters are preferable to casein promoters in the practice of the present invention because caseins generally are produced in female mammals during pregnancy as well as after birth, while WAP is expressed primarily during post-partum lactation. This difference is of potential importance for two reasons. First, pre-birth production of the desired protein under the transcriptional control of a casein promoter could be wasteful, since the protein cannot be isolated from milk until it is secreted into the milk post-partum. Second, where the desired protein is toxic in large amounts (human tissue plasminogen activator (t-PA) is an example), a build-up of the protein in the tissues prior to lactation could be deleterious to the health of the host mammal. An additional advantage of some whey promoters such as the WAP promoter is that they are strong promoters, as evidenced by the large amounts of some whey proteins present in milk. Casein promoters also have this advantage.

0009Milk protein genes from which promoters, in addition to the WAP promoters, can be isolated, can be obtained in the same manner in which the WAP genes were isolated, as described in Hennighausen and Sippel, id, and Campbell et al. (1984) Nucleic Acids Research 12, 8685. The method generally involves isolating the mRNA from a lactating mammary gland, constructing a cDNA library from the mRNA, screening the library for the particular milk protein cDNA being sought, cloning that cDNA into vectors, and using the appropriate cDNA as a probe to isolate the genomic clone from a genomic library. A sequence upstream from the transcription start site in the genomic clone constitutes a putative "promoter", a genomic sequence preceeding the gene of interest and presumed to be involved in its regulation. The promoter may be isolated by carrying out restriction endonuclease digestions and subcloning steps. Promoters need not be of any particular length nor to have directly shown any properties of regulation. The mouse WAP promoter was isolated as a 2.6 kb EcoRI - Kpnl fragment immediately 5' to the WAP signal sequence.

Desired

Protein

0010Any desired protein can be produced according to the invention. Preferred proteins are proteins useful in the treatment, prevention, and/or diagnosis of human disease; examples are t-PA and hepatitis B surface antigen. The invention is particularly useful for proteins which must be produced on a large scale to be economical, e.g., industrial enzymes and animal proteins.

Signal

Sequence

0011It is necessary, for secretion of the desired protein into the milk of the host mammal, that the DNA sequence containing the gene for the desired protein include DNA which, when translated, causes the protein to be secreted out of the mammary tissue into the milk. Without such a sequence, the desired protein would remain in the mammary tissue, from which purification would be difficult, and would require sacrifice of the host animal. This DNA can encode a hydrophobic secretion signal which is cleaved during secretion. The signal sequence can be that which is naturally associated with the desired protein, if the protein is normally secreted (e.g., t-PA). Alternatively, the signal encoding sequence can be that of the milk protein providing the promoter, i.e., when the milk protein gene is digested and the promoter isolated, a DNA fragment is selected which includes both the promoter and the signal encoding sequence directly downstream from the promoter. Another alternative is to employ a signal encoding sequence derived from another secreted protein, which is neither the milk protein normally expressed from the promoter nor the desired protein.

Termination

Site

0012Preferably there is located within or downstream from the 3' end of the desired gene a termination site. This site may be provided by sequences in the gene itself, or may need to be added. If the sequence is to be added, a preferred sequence is provided by the polyadenylation sequence of the virus SV40, as will be described in greater detail below.

Methods

Genetic Manipulations

0013Generally, all DNA manipulations used in the genetic constructions described herein may be carried out using conventional techniques, as described, e.g., in Maniatis et al. MolecularCloning:A LaboratoryManual (Cold Spring Harbor Laboratory, 1982).

Introduction

DNA

into

Embryos

0014Once the genetic constructions have been produced in vectors, e.g., plasmids, the promoter-signal sequence-desired protein-termination sequence DNA fragment is excised and then introduced into the desired mammalian embryo using, e.g., retroviruses or standard microinjection methods such as are described in Kraemer et al. (1985), Costantini and Jaenisch, eds., Genetic ManipulatonoftheEarlyMammalianEmbryo, Cold Spring Harbor Laboratory (bovine embryo microinjection); Hammer et al. (1985) Nature 315, 680 (rabbit, sheep, and porcine embryo microinjection); and Gordon and Ruddle (1984) Methods in Embryology 101, 411 (mouse embryo microinjection). Micro-injection is preferably carried out on an embryo at the one-cell stage, to maximize both the chances that the injected DNA will be incorporated into all cells of the animal, including mammary tissue, and that the DNA will also be incorporated into the germ cells, so that the animal's offspring will be transgenic as well. Microinjection is a standard technique which involves, briefly, isolating fertilized ova, visualizing the pronucleus, and then injecting the DNA into the pronucleus by holding the ova with a blunt holding pipette of a diameter on the order of 50 µm, and using a sharply pointed pipette of a diameter on the order of 1.5 µm to inject buffer-containing DNA into the pronucleus. Following microinjection, the transgenic female animals are allowed to become sexually mature, mated, and milk collected post-partum.

0015Preferred host mammals are those which are already bred for large volume milk production, e.g., cows, sheep, goats, and pigs.

t-PA Production

0016There will now be described the construction of plasmid DNA in which the gene encoding human uterine t-PA, including the signal encoding sequence, is under the transcriptional control of the mouse WAP promoter, and has at its 3' end the SV40 polyadenylation site. This DNA was made from two intermediate plasmids, one carrying the mouse WAP promoter and one carrying the t-PA signal and structural sequences, as well as the SV40 polyadenylation site.

0017The WAP promoter containing plasmid pWAP-CAT (Fig. 2, obtained from Lothar Hennighausen, National Institutes of Health) was derived from a plasmid made according to the methods described in Hennighausen and Sippel (1982) Eur. J. Biochem. 125, 131; and Campbell et al. (1984) Nucleic Acids Research 12, 8685. In addition to containing the mouse WAP promoter, pWAP-CAT contains a gene which, for present purposes, is irrelevant: the CAT (chloramphenical acetyltransferase) gene, which does not form a part of the final DNA sequence which is microinjected.

0018Still referring to Fig. 2, pWAP-CAT was modified to convert the EcoRI site to a HindIII site using Klenow and HindIII linkers.

0019The t-PA-containing plasmid pt-PA-VPl-LP(K) (Fig. 1) was derived from pt-PAVPI-LP, containing the t-PA gene (including the t-PA signal encoding sequence) and SV40 polyadenylation site, by modifying the unique NcoI site at the 5'end of the t-PA gene using NcoI endonuclease and Klenow and adding Kpn linkers to produce a KPnI site.

0020Referring to Fig. 3, the KpnI-BamHI fragment of pt-PA VP1-LP(K), containing the t-PA gene and SV40 sequences, was isolated and ligated to BamHI-KpnI treated pWAP(H3) to form pWAP-tPA (S), which was then transformed into a TET-sensitive derivative of E. coli strain MC1061. This transformed strain, containing plasmid DNA in which the HindIII-BamHI fragment contains the t-PA gene including the t-PA signal encoding sequence under the transcriptional control of the WAP promoter and followed by the SV40 polyadenylation site, has been deposited in the American Type Culture Collection on March 13, 1986 and given ATCC Accession No. 67032.

0021Production of milk into which t-PA has been secreted is carried out by excising the HindIII-BamHI fragment from the deposited strain and transferring it by microinjection or other means preferably into the one-cell embryo of a mammal according to conventional methods, as described above. Alternatively, though less desirably, the entire plasmid or other restriction fragments can be introduced into the embryos. Embryos are then nurtured to term invivo. Animals born from such manipulated embryos are screened for the presence of introduced DNA in the genome, and expression of t-PA in the milk is screened for among transgenic, lactating females. The protein from the milk of the adult lactating female animal will be assayed for t-PA by conventional procedures.

Production of Hepatitis B Surface Antigen

0022Referring to Figure 5, intermediate vectors pWAP-CAT and pHBsSVA were used to construct pWAP-Hbs(S), containing the gene for hepatitis B surface antigen, under the transcriptional control of the WAP promoter and followed by the SV40 polyadenylation site.

0023The plasmid pWAP-CAT is described above. Plasmid pHbsSVA was constructed as illustrated in Fig. 4. pCLH3A, containing the SV40 polyadenylation sequence, was restricted with EcoRI, SacI, and BglII. pBSBam, containing the gene for hepatitis B surface antigen, was cut with EcoRI, BamHI and PvuI, and the two mixtures ligated to give pHbsSVA, in which the SV40 sequence was positioned at the 3' end of the Hbs gene, on a BamHI-BglII fragment. This fragment was then ligated (Fig. 5) to BamHI and bacterial alkaline phosphatase-treated pWAP-CAT, transformed into E. coli strain MC1061, and the plasmid pWAP-Hbs(S) isolated.

0024The BamHI-EcoRI fragment of WAP-Hbs(S) can be excised and used as described above to produce hepatitis B surface antigen. Alternatively, though less desirably, the entire plasmid or other restriction fragments can be introduced into the embryos. Embryos are then nurtured to term invivo. Animals born from such manipulated embryos are screened for the presence of introduced DNA in the genome, and expression of hepatitis B surface antigen in the milk is screened for among transgenic, lactating females. pWAP-Hbs(S) has been deposited in the American Type Culture Collection on March 13, 1986 and given ATCC Accession No. 67033. Applicants' assignee, Integrated Genetics, Inc.,

0025Both pWAP-Hbs(S) and pWAP-t-PA(S) can be used as cassette vectors in which the hepatitis B surface antigen gene or the t-PA gene can be excised and replaced, using conventional methods, with any desired gene. If desired, the signal encoding sequence from pWAP-t-PA(S) can be left in the vector, and a gene lacking such a sequence inserted downstream of and in frame with it. Alternatively, the signal sequence from pWAP-t-PA(S) or pWAP-Hbs(S) can be removed along with the structural gene and the signal encoding sequence of the substituted gene employed. In addition, the WAP promoter alone can be excised and inserted into another desired expression vector.

Purification and Use

0026The proteins produced by a process according to the invention are purified from the milk into which they have been secreted and used for their known purposes.

0027Hepatitis B surface antigen is useful in the production of hepatitis B vaccine.

0028t-PA is useful in the treatment of thrombolytic disease in which fibrin clot lysis is necessary, as described in European Patent Application 85306957.3. That Patent Application also describes general purification techniques which will be useful for milk-secreted proteins.

Stability in Milk

0029Table I below shows that, despite the presence in milk of numerous proteases, recombinant t-PA is stable when added to raw goat milk and incubated at 20° or 37°C for 24 hours, with no evidence of loss of activity, as measured using the standard fibrin plate test (results not shown in Table I) or the amidolytic assay described in Wei et al., id. Similarly, recombinant hepatitis B surface antigen was found to be stable for at least 24 hours in raw goat milk (data not shown). <tables id="tabl0001" num="0001"><table frame="all"><title>Table I</title><tgroup cols="4" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="39.37mm" /><colspec colnum="2" colname="col2" colwidth="39.37mm" /><colspec colnum="3" colname="col3" colwidth="39.37mm" /><colspec colnum="4" colname="col4" colwidth="39.37mm" /><thead valign="top"><row><entry namest="col1" nameend="col4" align="center">Amidolytic assay for TPA</entry></row><row><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" align="center">Incubation Time</entry><entry namest="col3" nameend="col3" align="center">Temperature</entry><entry namest="col4" nameend="col4" align="center">Units/ml</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">Goat milk alone</entry><entry namest="col2" nameend="col2" align="center">--</entry><entry namest="col3" nameend="col3" align="center">--</entry><entry namest="col4" nameend="col4" align="center"><20, <20</entry></row><row><entry namest="col1" nameend="col1" align="left">Goat milk & TPA</entry><entry namest="col2" nameend="col2" align="center">0</entry><entry namest="col3" nameend="col3" align="center">--</entry><entry namest="col4" nameend="col4" align="center">437, 368</entry></row><row><entry namest="col1" nameend="col1" align="left">Goat milk & TPA</entry><entry namest="col2" nameend="col2" align="center">24 hours</entry><entry namest="col3" nameend="col3" align="center">20°C</entry><entry namest="col4" nameend="col4" align="center">419, 434</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">Goat milk & TPA</entry><entry namest="col2" nameend="col2" align="center">24 hours</entry><entry namest="col3" nameend="col3" align="center">37°C</entry><entry namest="col4" nameend="col4" align="center">467, 507</entry></row></tbody></tgroup></table></tables>

Other Embodiments

0030Other embodiments are feasible. For example, other milk serum protein promoters can be used in place of the mouse WAP promoter, and the promoter can be derived from any mammalian species. For example, milk serum protein promoters such as that of β-lactoglobulin can be used, and the rat, rather than mouse, WAP promoter can be used; the rat WAP promoter is described in Campbell et al., id. Although less desirable than milk serum protein promoters, casein promoters can be used as well. The protein produced using processes in accordance with this invention can be any desired protein of therapeutic or industrial importance.

Contents2

Every citation, both ways

Citations
Document	Relation	Office	Cited during
US7939317B1	Cited by	United States of America	Applicant
US5487992A	Cited by	United States of America	Search report
US6743966B2	Cited by	United States of America	Applicant
US6268545B1	Cited by	United States of America	Applicant
EP0527063A1	Cited by	European Patent Office (EPO)	Search report
US6746852B1	Cited by	United States of America	Applicant
US12275939B2	Cited by	United States of America	Applicant
US12331295B2	Cited by	United States of America	Applicant
US12384828B2	Cited by	United States of America	Applicant
EP0451823A3	Cited by	European Patent Office (EPO)	Search report
US5831141A	Cited by	United States of America	Search report
US4873316A	Cited by	United States of America	Search report
US5994616A	Cited by	United States of America	Search report
US8980626B2	Cited by	United States of America	Applicant
US10975354B2	Cited by	United States of America	Applicant
US5476995A	Cited by	United States of America	Search report
US5322775A	Cited by	United States of America	Search report
US5739407A	Cited by	United States of America	Search report
US8530629B2	Cited by	United States of America	Applicant
EP0279582A3	Cited by	European Patent Office (EPO)	Search report
US7833727B2	Cited by	United States of America	Applicant
US6232094B1	Cited by	United States of America	Applicant
US5895833A	Cited by	United States of America	Search report
US6222094B1	Cited by	United States of America	Applicant
US7097993B2	Cited by	United States of America	Applicant
AU628101B2	Cited by	Australia	Search report
EP0527063A1	Cited by	European Patent Office (EPO)	Search report
US9494597B2	Cited by	United States of America	Applicant
US5589604A	Cited by	United States of America	Search report
WO9005188A1	Cited by	World Intellectual Property Organization (WIPO)	International search
EP0279582A2	Cited by	European Patent Office (EPO)	Search report
EP0959907A1	Cited by	European Patent Office (EPO)	Search report
US6448469B1	Cited by	United States of America	Search report
US6733991B1	Cited by	United States of America	Applicant
US6348349B1	Cited by	United States of America	Applicant
WO9325567A1	Cited by	World Intellectual Property Organization (WIPO)	International search
US6262336B1	Cited by	United States of America	Applicant
FR2677652A1	Cited by	France	Search report
US6448469B1	Cited by	United States of America	Applicant
US5776773A	Cited by	United States of America	Search report
EP0347431A1	Cited by	European Patent Office (EPO)	Search report
US5861299A	Cited by	United States of America	Search report
US5895833A	Cited by	United States of America	Search report
US9624290B2	Cited by	United States of America	Applicant
US6548735B1	Cited by	United States of America	Applicant
US5965788A	Cited by	United States of America	Search report
US11976106B2	Cited by	United States of America	Applicant
US10669562B2	Cited by	United States of America	Applicant
AU656720B2	Cited by	Australia	Search report
US5366894A	Cited by	United States of America	Search report
US6689610B1	Cited by	United States of America	Applicant
US5464764A	Cited by	United States of America	Search report
EP0347431A4	Cited by	European Patent Office (EPO)	Search report
US9879297B2	Cited by	United States of America	Applicant
US5965789A	Cited by	United States of America	Search report
WO9315196A1	Cited by	World Intellectual Property Organization (WIPO)	International search
WO9004036A1	Cited by	World Intellectual Property Organization (WIPO)	International search
US7329504B2	Cited by	United States of America	Applicant
WO9004036A1	Cited by	World Intellectual Property Organization (WIPO)	International search
US9206248B2	Cited by	United States of America	Applicant
US8663980B2	Cited by	United States of America	Applicant
US5650503A	Cited by	United States of America	Search report
WO9005188A1	Cited by	World Intellectual Property Organization (WIPO)	International search
WO9222644A1	Cited by	World Intellectual Property Organization (WIPO)	International search
US5304489A	Cited by	United States of America	Search report
US7288385B2	Cited by	United States of America	Applicant
US10478475B2	Cited by	United States of America	Applicant
EP0451823A2	Cited by	European Patent Office (EPO)	Search report
US7494784B2	Cited by	United States of America	Applicant
US6472584B1	Cited by	United States of America	Applicant
US5565362A	Cited by	United States of America	Search report
US8334111B2	Cited by	United States of America	Applicant
US4873316A	Cites	United States of America	Examiner
EP0169672A	Cites	European Patent Office (EPO)	–
US4873316A	Cites	United States of America	–
NATURE, vol. 315, 1985; LOVEIL-BADGE, pp. 628-629	Non-patent	–	–
JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 260, no. 11, 1985; JONES et al., pp. 7042-50	Non-patent	–	–
BIO/TECHNOLOGY, vol. 5, 1987; GORDON et al., pp. 1183-1187	Non-patent	–	–
BIO/TECHNOLOGY, vol. 9, 1991; EBERT et al., pp. 835-849	Non-patent	–	–
NATURE, vol. 313, 14 February 1985, New York-London; D.M. ORNITZ et al., pp. 600-602	Non-patent	–	–
NATURE, vol. 315, 23 May 1985, New York-London; J. MAGRAM et al., pp. pp. 338-340	Non-patent	–	–
NATURE, vol. 300, no. 5893, 16 December 1982, New York-London; R.D. PALMITER et al., pp. 611-615	Non-patent	–	–
MOLECULAR & CELLULAR BIOLOGY, vol. 5, no. 7, July 1985, Washington, DC (US); R. KRUMLAUF et al., pp. 1639-1648	Non-patent	–	–
EUROPEAN JOURNAL OF BIOCHEMISTRY, vol. 125, no. 1, 02 June 1982, Berlin-Heidelberg-New York; L.G. HENNIGHAUSEN et al., pp. 131-141	Non-patent	–	–
NATURE, vol. 315, 1985; LOVEIL-BADGE, pp. 628-629	Non-patent	–	Examiner
JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 260, no. 11, 1985; JONES et al., pp. 7042-50	Non-patent	–	Examiner
BIO/TECHNOLOGY, vol. 5, 1987; GORDON et al., pp. 1183-1187	Non-patent	–	Examiner
BIO/TECHNOLOGY, vol. 9, 1991; EBERT et al., pp. 835-849	Non-patent	–	Examiner
NATURE, vol. 315, 23 May 1985, New York-London; J. MAGRAM et al., pp. pp. 338-340	Non-patent	–	Examiner
EUROPEAN JOURNAL OF BIOCHEMISTRY, vol. 125, no. 1, 02 June 1982, Berlin-Heidelberg-New York; L.G. HENNIGHAUSEN et al., pp. 131-141	Non-patent	–	Examiner

16 members in 7 offices; this record represents the family

This recordMember, by publication date

Members16

Family members, oldest first
Document	Office	Kind
JPS63291A	Japan	A	1988-01-05
EP0264166A1	European Patent Office (EPO)	A1	1988-04-20
EP0264166B1This record	European Patent Office (EPO)	B1	1996-08-21
AT141646T	Austria	T	1996-09-15
DE3751873D1	Germany	D1	1996-09-26
DE3751873T2	Germany	T2	1997-02-13
JPH09294586A	Japan	A	1997-11-18
JP2874751B2	Japan	B2	1999-03-24
US6727405B1	United States of America	B1	2004-04-27
US7045676B1	United States of America	B1	2006-05-16
LU91304I2	Luxembourg	I2	2007-03-12
NL300257I1	Netherlands (Kingdom of the)	I1	2007-04-02
DE122007000007I1	Germany	I1	2007-05-16
NL300257I2	Netherlands (Kingdom of the)	I2	2007-06-01
DE122007000007I2	Germany	I2	2010-12-30
US7939317B1	United States of America	B1	2011-05-10

81 legal events, as 10 offices reported them to INPADOC

Over the term

Point at a mark for the event

Events

Legal events, newest first
	Event	Code	Office
2012-05-02	Supplementary protection certificate expiredExpiredSPCE	SPCE	GB
2012-04-18	Supplementary protection certificate expiredExpiredSPCX	SPCX	NL
2012-04-18	Supplementary protection certificate expiredExpiredSPCX	SPCX	NL
2012-04-10	Expiry of right [spc]SP71	SP71	DE
2011-11-23	Supplementary protection certificate entered into forceCTFE	CTFE	GB
2011-08-24	Supplementary protection certificate grantedGrantedCTFG	CTFG	GB
2010-10-31	Expiry of a supplementary protection certificateCCRE	CCRE	BE
2010-09-07	Supplementary protection certificate grantedGrantedR069	R069	DE
2010-07-28	Decision to grant supplementary protection certificate by patent divisionR067	R067	DE
2008-03-18	Supplementary protection certificate grantedGrantedSPCG	SPCG	SE
2008-03-18	Supplementary protection certificate grantedGrantedSPCG	SPCG	SE
2007-12-31	Be: patent expiredExpiredBE20	BE20	EP
2007-12-28	Other action affecting the existence or the validity of an industrial property rightAV	AV	FR
2007-12-28	Other action affecting the existence or the validity of an industrial property rightAV	AV	FR
2007-11-24	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2007-10-26	Supplementary certificate of protection granted (eec regulation of 18 june 1992)GrantedCY	CY	FR
2007-08-15	Grant for a certificate of protection (e-series)EEZF	EEZF	AT
2007-07-15	Cancelled due to lapse of timeLapsedEELA	EELA	AT
2007-06-19	Se: european patent has lapsedLapsedEUG	EUG	EP
2007-06-01	Grant of a supplementary protection certificateKC1	KC1	NL
2007-06-01	Nl: ceased due to reaching the maximum lifetime of a patentCeasedNLV7	NLV7	EP
2007-05-15	Patent ceasedCeasedPL	PL	CH
2007-05-11	Supplementary protection certificate (spc) filedCP	CP	FR
2007-05-02	Patent expired after termination of 20 yearsExpiredPE20	PE20	GB
2007-04-09	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2007-04-02	Application for a supplementary protection certificateAC1	AC1	NL
2007-03-15	Application filed for a certificate of protection (e-series)ESZA	ESZA	AT
2007-03-12	Supplementary protection certificateCCP	CCP	LU
2007-03-09	Request for supplementary protection certificate laid open to the public (eec regulation of 18 june 1992)CR	CR	FR
2007-03-08	Application for supplementary protection certificate effectively filedR065	R065	DE
2007-02-20	Application for supplementary protection certificate filedSPCF	SPCF	SE
2007-02-20	Application for supplementary protection certificate filedSPCF	SPCF	SE
2007-02-07	Supplementary protection certificate filedCTFF	CTFF	GB
2007-01-25	Supplementary protection certificate (spc) filedCP	CP	FR
2006-05-31	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2006-05-17	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2006-05-16	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2006-05-03	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2006-04-30	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2006-04-26	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2006-04-26	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2006-04-24	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2006-04-17	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2006-03-21	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2002-07-01	Nl: decision of oppositionOppositionNLR2	NLR2	EP
2002-04-17	Opposition proceedings terminatedOpposition27C	27C	EP
2002-03-02	Termination of opposition procedure: date of legal effect publishedOppositionORIGINAL CODE: 0009276PLBM	PLBM	EP
2002-03-02	Information on the status of an ep patent application or granted ep patentGrantedSTATUS: OPPOSITION PROCEDURE CLOSEDSTAA	STAA	EP
2002-01-01	European patent in force as of 2002-01-01IF02	IF02	GB
2001-09-25	Opposition procedure terminatedOppositionORIGINAL CODE: EPIDOS OPPCPLBL	PLBL	EP
1998-10-20	Observations filed by third partiesORIGINAL CODE: EPIDOS TIPATPAD	TPAD	EP
1998-07-22	Reply of patent proprietor to notice(s) of oppositionOppositionORIGINAL CODE: EPIDOS OBSOPLBF	PLBF	EP
1998-04-29	Reply of patent proprietor to notice(s) of oppositionOppositionORIGINAL CODE: EPIDOS OBSOPLBF	PLBF	EP
1998-03-02	Reply of patent proprietor to notice(s) of oppositionOppositionORIGINAL CODE: EPIDOS OBSOPLBF	PLBF	EP
1998-02-20	Reply of patent proprietor to notice(s) of oppositionOppositionORIGINAL CODE: EPIDOS OBSOPLBF	PLBF	EP
1997-11-20	Reply of patent proprietor to notice(s) of oppositionOppositionORIGINAL CODE: EPIDOS OBSOPLBF	PLBF	EP
1997-09-01	Nl: opposition has been filed with the epoOppositionNLR1	NLR1	EP
1997-07-09	Opposition filedOpposition26	26	EP
1997-06-26	Reply of patent proprietor to notice(s) of oppositionOppositionORIGINAL CODE: EPIDOS OBSOPLBF	PLBF	EP
1997-05-23	Opposition filedOppositionORIGINAL CODE: 0009260PLBI	PLBI	EP
1997-05-23	Unpublished change to opponent dataORIGINAL CODE: EPIDOS OPPOPLBQ	PLBQ	EP
1997-03-15	Publication of translation of european patent specificationUEP	UEP	AT
1996-11-18	It: translation for a ep patent filedITF	ITF	EP
1996-11-15	Fr: translation filedET	ET	EP
1996-10-31	New agentNV	NV	CH
1996-09-26	Corresponds to:REF	REF	EP
1996-08-21	Designated contracting statesAK	AK	EP
1996-08-21	Corresponds to:REF	REF	EP
1996-07-10	Licence recorded950526 0100 PHARMACEUTICAL PROTEINS LIMITED * 960404 0200 GENZYME TRANSGENICS CORPORATION111L	111L	EP
1996-07-05	(expected) grantORIGINAL CODE: 0009210GRAA	GRAA	EP
1995-07-26	Licence recorded0100 PHARMACEUTICAL PROTEINS LIMITED111L	111L	EP
1994-05-04	Party data changed (applicant data changed or rights of an application transferred)RAP1	RAP1	EP
1994-02-16	Nl: translation of patent claims filedTCNL	TCNL	EP
1992-12-30	Information on inventor provided before grant (corrected)RIN1	RIN1	EP
1992-12-30	Information on inventor provided before grant (corrected)RIN1	RIN1	EP
1992-12-30	Information on inventor provided before grant (corrected)RIN1	RIN1	EP
1992-12-30	Information on inventor provided before grant (corrected)RIN1	RIN1	EP
1992-05-13	First examination report despatched17Q	17Q	EP
1988-05-18	Request for examination filed17P	17P	EP
1988-04-20	Designated contracting statesAK	AK	EP
1988-03-05	Public reference made under article 153(3) epc to a published international application that has entered the european phaseORIGINAL CODE: 0009012PUAI	PUAI	EP

Transgenic animals secreting desired proteins into milk

Abstract

Term

DNA

DNA

Members16

Over the term

Events

Numbers

Titles3

Classification

Designated states11