Claims for Patent: 8,030,538

Summary for Patent: 8,030,538

Title:	Cattle beta-casein gene targeting vector using homologous recombination
Abstract:	The present invention relates to a bovine beta-casein gene targeting vector comprising (1) a first region having a length of 5 to 12 kb which is homologous to the promoter and its flanking nucleic acid sequences of bovine beta-casein gene, and comprising exon 1, intron 1, and exon 2 of bovine beta-casein gene; (2) a region for cloning a nucleic acid coding for desired proteins; (3) a region for coding a positive selection marker; (4) a second region having a length of 2.8 to 3.5 kb which is homologous to the nucleic acid sequences of bovine beta-casein gene, and comprising exon 5, 6, 7 and 8, and intron 5, 6 and 7 of bovine beta-casein gene; wherein the nucleic acid segment corresponding to the first region is located upstream to the nucleic acid segment corresponding to the second region in the 5\'-3\' arrangement of beta-casein gene. The present invention also relates to method producing the transgenic cattle which is bovine beta-casein gene-targeted with a gene coding a desired protein using the said vector and obtaining a large scale of a desired protein from the milk of the said transgenic cattle.
Inventor(s):	Han; Yong-Mahn (Daejeon, KR), Lee; Kyung-Kwang (Daejeon, KR), Chang; Mira (Gyeongsangbuk-Do, KR), Koo; Deog-Bon (Daejeon, KR)
Assignee:	Korea Research Institute of Bioscience and Biotechnology (Daejeon, KR)
Application Number:	10/574,747
Patent Claims:	1. A bovine beta-casein gene targeting vector comprising, in operable association, (1) a first nucleic acid region having a length of about 6 kb which comprises the promoter and its flanking nucleic acid sequences of a bovine beta-casein gene, and further comprises exon 1, intron 1, and exon 2 of a bovine beta-casein gene; (2) a region for cloning a nucleic acid coding for desired proteins; (3) a region for coding a positive selection marker; (4) a second nucleic acid region having a length of 2.8 to 3.5 kb which comprises exon 5, 6, 7 and 8, and intron 5, 6 and 7 of bovine beta-casein gene; wherein the nucleic acid segment corresponding to the first region is located upstream to the nucleic acid segment corresponding to the second region in the 5' -3' arrangement of beta-casein gene. 2. The vector according to claim 1, wherein the length of the second region is 3.0 to 3.2 kb. 3. The vector according to claim 1, wherein the positive selection marker is selected from the group consisting of neomycin (Neo) , hygromycin (Hyg) , histidmol dehydrogenase gene (hisD) and guanine phosphosribosyltransferase (Gpt). 4. The vector according to claim 1, wherein the vector further comprises a region for a negative selection marker. 5. The vector according to claim 4, wherein the negative selection marker is Diphtheria toxin (DT) gene. 6. A method for producing a bovine beta-casein gene-targeted somatic cell which comprises the steps of (1) introducing the bovine beta-casein gene-targeting vector according to claim 1 or 4 into a bovine fibroblast cell; (2) permitting to occur homologous recombination events in the bovine fibroblast cell; and (3) selecting the bovine beta-casein gene-targeted bovine embryonic cell or fibroblast cell with a desired gene. 7. The method according to claim 6, wherein the vector in the step (1) is introduced in form of linearized or deleted form lacking plasmid vector backbone. 8. A method for generating transgenic bovine which comprises the steps of (1) introducing the bovine beta- casein gene-targeting vector according to claim 1 or 4 into a bovine fibroblast cell; (2) permitting to occur homologous recombination events in the bovine fibroblast cell; (3) selecting the bovine beta-casein gene-targeted fibroblast cell with a desired gene; (4) introducing the nucleus of the bovine gene-targeted fibroblast cell into a nuclear-removed bovine oocyte to produce a nuclear-transferred bovine embryo; (5) activating the embryo; (6) implanting the embryo into a female bovine recipient; and (7) permitting the implanted embryo to develop. 9. A method for obtaining a large scale of desired proteins which comprise the steps of (1) generating transgenic cattle in accordance with the method of claim 8; (2) inducing lactation in the transgenic bovine; (3) collecting milk from the lactating transgenic bovine; and (4) purifying the desired protein from milk of the transgenic cattle.

Details for Patent 8,030,538

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132	06/04/1986		2024-11-23
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2024-11-23
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2024-11-23