VEGF Receptor Conditional Knockout Animals and Methods of Use

• US 20070250941A1
• Filed: 03/26/2007
• Published: 10/25/2007
• Est. Priority Date: 03/27/2006
• Status: Abandoned Application

• Current Assignee: Genentech, Inc. (Roche Holding AG)
• Original Assignee: Genentech, Inc. (Roche Holding AG)
• Inventors: Ferrara, Napoleone, Gerber, Hans-Peter, Baldwin, Megan
• Application Number:
  US11/691,469
• Publication Number:
  US 20070250941A1
• Time in Patent Office: Days
• US Class Current: 800/14
• CPC Class Codes: A01K 2217/075 inducing loss of function, i.e. knock out
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/03 Animal model, e.g. for test or diseases
• CPC Class Codes: A01K 67/0276 Knock-out vertebrates
• CPC Class Codes: C07K 14/71 for growth factors; for growth regulators
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 2800/30 Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
• CPC Class Codes: C12N 2830/008 cell type or tissue specific enhancer/promoter combination

Abstract

Transgenic non-human animals and methods of producing non-human transgenic animals comprising within their genome a conditional targeted mutation in a VEGFR, e.g., VEGFR1 or VEGFR2, gene are provided along with methods and vectors used to produce such animals or cells.

Claims

1. A transgenic non-human animal comprising a conditional targeted VEGFR-1 mutation, wherein the conditional targeted mutation comprises one or both alleles of a VEGFR-1 gene.

2. The transgenic non-human animal of claim 1, wherein the conditional targeted VEGFR-1 mutation comprises recognition sites for a recombinase framing a genomic sequence of VEGFR-1 encoding a polypeptide with VEGFR-1 activity, wherein the genomic sequence comprises at least one exon of a VEGFR-1 gene.

3. The transgenic non-human animal of claim 2, wherein the at least one exon comprises exon 1 of the VEGFR-1 gene.

4. The transgenic non-human animal of claim 1, wherein the non-human animal is selected from the group consisting of a rodent, a rat, a rabbit, a monkey, a guinea pig, a dog, a sheep, a horse, a dog and a cat.

5. The transgenic non-human animal of claim 1, wherein the non-human animal is a mouse.

6. The transgenic non-human animal of claim 1, wherein the transgenic non-human mouse expresses no native expression of VEGFR-1 compared to a control transgenic non-human animal.

18. A method for determining VEGFR-1 function, the method comprising:

administering an inducer of conditional mutation of a VEGFR-1 gene to a conditional transgenic conditional targeted VEGFR-1 mutation non-human animal of claim 1, thereby producing an induced conditional transgenic non-human animal;

analysizing and comparing the induced conditional transgenic non-human animal with a non-induced conditional transgenic non-human animal; and

,

determining differences, thereby determining the function of VEGFR-1.

7. A transgenic cell comprising a conditional targeted VEGFR-1 mutation, wherein the conditional targeted mutation comprises one or both alleles of a VEGFR-1 gene.

8. A vector construct comprising a genomic sequence of VEGFR-1 gene, wherein the genomic sequence comprises at least one exon of a VEGFR-1 gene and a positive selection marker and wherein the genomic sequence is framed by recognition sites for recombinase.

9. The vector construct of claim 8, wherein the at least one exon comprises exon 1 of the VEGFR-1 gene.

10. The vector construct of claim 8, wherein the positive selection marker is selected from the group consisting of a neomycin resistance gene and a hygromycin resistance gene.

11. The vector construct of claim 8, wherein the genomic sequence further comprises a negative selection marker.

12. The vector construct of claim 11, wherein the negative selection marker is selected from the group consisting of a diphtheria toxin gene and an HSV-thymidine kinase gene (HSV-TK).

13. A host cell comprising the vector construct of claim 8.

14. A method of producing a transgenic conditional targeted VEGFR-1 mutation non-human animal, the method comprising:

introducing a vector construct of claim 8 into an embryonic stem cell of a non-human animal; and

generating a heterozygous and/or homozygous transgenic animal from the embryonic stem cell, thereby producing the transgenic conditional targeted VEGFR-1 mutation non-human animal.

15. The method of claim 14, further comprises cross breeding the transgenic conditional targeted VEGFR-1 mutation non-human animal with second transgenic animal.

16. The method of claim 15, wherein the second transgenic animal is a transgenic non-human animal with an inducer.

17. The method of claim 15, wherein the second transgenic animal is a transgenic conditional VEGF targeted mutation non-human animal.

19. A transgenic non-human animal comprising a conditional targeted VEGFR-2 mutation, wherein the conditional targeted mutation comprises one or both alleles of a VEGFR-2 gene.

20. The transgenic non-human animal of claim 19, wherein the conditional targeted VEGFR-2 mutation comprises recognition sites for a recombinase framing a genomic sequence of VEGFR-2 encoding a polypeptide with VEGFR-2 activity, wherein the genomic sequence comprises at least one exon of a VEGFR-2 gene.

21. The transgenic non-human animal of claim 20, wherein the at least one exon comprises exon 1 of the VEGFR-2 gene.

22. The transgenic non-human animal of claim 19, wherein the non-human animal is selected from the group consisting of a rodent, a rat, a rabbit, a monkey, a guinea pig, a dog, a sheep, a horse, a dog and a cat.

23. The transgenic non-human animal of claim 19, wherein the non-human animal is a mouse.

24. The transgenic non-human animal of claim 19, wherein the transgenic non-human animal expresses no native expression of VEGFR-2 compared to a control transgenic non-human animal.

36. A method for determining VEGFR-2 function, the method comprising:

administering an inducer of a conditional mutation of a VEGFR-2 gene to a conditional transgenic conditional targeted VEGFR-2 mutation non-human animal of claim 19, thereby producing an induced conditional transgenic non-human animal;

analysizing and comparing the induced conditional transgenic non-human animal with a non-induced conditional transgenic non-human animal; and

,

determining differences, thereby determining the function of VEGFR-2.

25. A transgenic cell comprising a conditional targeted VEGFR-2 mutation, wherein the conditional targeted mutation comprises one or both alleles of a VEGFR-2 gene.

26. A vector construct comprising a genomic sequence of VEGFR-2 gene, wherein the genomic sequence comprises at least one exon of a VEGFR-2 gene and a positive selection marker and wherein the genomic sequence is framed by recognition sites for recombinase.

27. The vector construct of claim 26, wherein the at least one exon comprises exon 1 of the VEGFR-2 gene.

28. The vector construct of claim 26, wherein the positive selection marker is selected from the group consisting of a neomycin resistance gene and a hygromycin resistance gene.

29. The vector construct of claim 26, wherein the genomic sequence further comprises a negative selection marker.

30. The vector construct of claim 29, wherein the negative selection marker is selected from the group consisting of a diphtheria toxin gene and an HSV-thymidine kinase gene (HSV-TK).

31. A host cell comprising the vector construct of claim 26.

32. A method of producing a transgenic conditional targeted VEGFR-2 mutation non-human animal, the method comprising:

introducing a vector construct of claim 26 into an embryonic stem cell of a non-human animal; and

generating a heterozygous and/or homozygous transgenic animal from the embryonic stem cell, thereby producing the transgenic conditional targeted VEGFR-2 mutation non-human animal.

33. The method of claim 32, further comprises:

cross breeding the transgenic conditional targeted VEGFR-2 mutation non-human animal with second transgenic animal.

34. The method of claim 33, wherein the second transgenic animal is a transgenic non-human animal with an inducer.

35. The method of claim 33, wherein the second transgenic animal is a transgenic conditional VEGF targeted mutation non-human animal.

37. A transgenic non-human animal comprising a conditional targeted VEGFR mutation, wherein the conditional targeted mutation comprises one or both alleles of a VEGFR gene.

38. A transgenic non-human animal comprising a VEGFR-1-Cre transgenic non-human animal.

39. A transgenic non-human animal comprising a conditional VEGFR-1-loxP transgenic non-human animal.

40. A transgenic non-human animal comprising a conditional a VEGFR-2 loxP transgenic non-human animal.

41. A transgenic non-human animal comprising a VEGFR-1-Cre:

VEGF-loxP transgenic non-human animal.