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Last Updated: March 29, 2024

Claims for Patent: 6,653,113


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Summary for Patent: 6,653,113
Title: High efficiency gene targeting in mouse embryonic stem cells
Abstract:The present invention provides novel methods for modifying the genome of an animal cell which typically comprise the steps of: constructing a DNA molecule in which desired sequence modifications are contained in a segment of DNA (a \"targeting DNA\") that is substantially isogenic with a DNA in the cell genome (a \"target DNA\"); introducing the targeting DNA construct into the cell (e.g., by microinjection, electroporation, transfection, or calcium phosphate precipitation); and selecting cells in which the desired sequence modifications have been introduced into the genome via homologous recombination.
Inventor(s): Berns; Anton (Spaarndam, NL), Robanus Maandag; Els (Haarlem, NL), te Riele; Hein (Amsterdam, NL)
Assignee: Genpharm International, Inc. (Mountain View, CA)
Application Number:09/253,818
Patent Claims:1. A method for modifying a target DNA sequence in a mouse embryonic stem cell comprising: (a) introducing in vitro a targeting DNA sequence into the mouse embryonic stem cell derived from an inbred mouse strain, said targeting DNA sequence is isolated from said inbred mouse strain; and (b) isolating in vitro the mouse embryonic stem cell whose target DNA sequence has been modified by incorporation of said targeting DNA sequence into a nonselectable gene of the target sequence.

2. The method of claim 1 wherein said inbred mouse strain is 129.

3. The method of claim 1 wherein said inbred mouse strain is BALB/c.

4. The method of claim 1 wherein said mouse embryonic stem cell is derived from a substrain of said inbred mouse strain.

5. The method of claim 1 wherein said targeting DNA sequence is isolated from a substrain of said inbred mouse strain.

6. The method of claim 1 wherein said mouse embryonic stem cell is derived from a first substrain of said inbred mouse strain and wherein said targeting DNA sequence is isolated from a second substrain of said inbred mouse strain.

7. The method of claim 6 wherein said first substrain and said second substrain are the same substrain.

8. The method of claim 1 wherein said targeting DNA sequence, except for desired sequence modifications, is at least about 99.5% identical with said target DNA sequence in the mouse embryonic stem cell.

9. The method of claim 1 wherein said targeting DNA sequence, except for desired sequence modifications, is at least about 99.9% identical with said target DNA sequence in the mouse embryonic stem cell.

10. The method of claim 1 wherein said targeting DNA sequence contains a selectable marker gene.

11. The method of claim 10 wherein said selectable marker gene is a gene conferring resistance to a compound inhibitory to cell growth.

12. The method of claim 10 wherein said selectable marker gene is a gene conferring the ability to grow on a selected substrate.

13. The method of claim 10 wherein said selectable marker gene is a neomycin resistance gene.

14. The method of claim 10 wherein said selectable marker gene lacks its own promoter.

15. The method of claim 10 wherein said selectable marker gene has no poly(A) sequence.

16. The method of claim 10 wherein said selectable marker gene is placed in an intron.

17. The method of claim 1 wherein said target DNA sequence has been modified by a replacement-type event.

18. The method of claim 1 wherein said target DNA sequence has been modified by an insertion-type event.

19. The method of claim 1 wherein said targeting DNA sequence is part of a DNA delivery molecule which contains additional DNA sequence flanking the targeting DNA sequence.

20. The method of claim 19 wherein said additional DNA sequence contains a selectable marker.

21. The method of claim 1 wherein said targeting DNA sequence, except for desired sequence modifications, is at least about 300 base pairs.

22. The method of claim 1 wherein said targeting DNA sequence, except for desired sequence modifications, is at least about 1000 base pairs.

23. The method of claim 1 wherein said targeting DNA sequence is introduced into said mouse embryonic stem cell by a method selected from the group consisting of microinjection, electroporation, calcium phosphate precipitation, liposome fusion and transfection using a virus or a viral particle.

Details for Patent 6,653,113

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 ⤷  Try a Trial 2011-08-20
Merck Sharp & Dohme Corp. INTRON A interferon alfa-2b For Injection 103132 ⤷  Try a Trial 2011-08-20
Merck Sharp & Dohme Corp. INTRON A interferon alfa-2b Injection 103132 ⤷  Try a Trial 2011-08-20
>Applicant >Tradename >Biologic Ingredient >Dosage Form >BLA >Approval Date >Patent No. >Expiredate

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