Transgenic animal with recombinant vascular endothelial growth factor B (VEGF-B DNA) and uses thereof

• US 6,262,337 B1
• Filed: 02/18/1998
• Issued: 07/17/2001
• Est. Priority Date: 02/18/1997
• Status: Expired due to Fees

• Current Assignee: Ludwig Institute for Cancer Research, Ltd.
• Original Assignee: Ludwig Institute for Cancer Research, Ltd.
• Inventors: Li, Xuri, Aase, Karin, Pekny, Milos, Betsholtz, Christer, von Euler, Gabriel, Gebre-Medhin, Samuel, Eriksson, Ulf
• Primary Examiner(s): Hauda, Karen M.
• Application Number:
  US09/025,701
• Time in Patent Office: 1,245 Days
• Field of Search: 800/3, 800/8, 800/18, 800/21, 800/25, 800/24, 800/22, 435/325, 435/335, 435/354, 435/375, 424/93.21
• US Class Current: 800/18
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• 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 67/0271 Chimeric vertebrates, e.g. comprising exogenous cells
• CPC Class Codes: A01K 67/0276 Knock-out vertebrates
• CPC Class Codes: C07K 14/52 Cytokines; Lymphokines; Interferons
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic

Abstract

Transgenic animals containing recombinant DNA with modified nucleotide sequence from the vascular endothelial growth factor B (VEGF-B) gene, cells and methods for producing such animals, and methods of using them to assay substances for VEGF-B-like activity.

Claims

1. A transgenic mouse whose genome comprises a homozygous disruption in the endogenous VEGF-B gene, wherein said disruption in the endogenous VEGF-B gene is caused by the insertion of a DNA sequence into said endogenous VEGF-B gene, and wherein said disruption results in the lack of expression of VEGF-B and wherein said transgenic mice are viable.

2. The transgenic mouse of claim 1, wherein the disrupted VEGF-B gene is disrupted by point mutation, site-directed mutagenesis, deletion, substitution, or insertion.

3. The transgenic mouse of claim 2, wherein the disrupted VEGF-B gene has at least one codon for cysteine changed or deleted.

4. The transgenic mouse of claim 1, wherein the heart of said mouse is smaller than that of a wild-type mouse of the same species.

5. The transgenic mouse of claim 1, wherein the growth of an implanted tumor is slower than the growth of the same tumor in a wild-type mouse of the same species.

6. A method for producing the transgenic mouse of claim 1, comprising:

(a) introducing into a mouse embryonic cell a DNA sequence encoding a selectable marker and having a 5′

region and a 3′

region of homology to the genomic VEGF-B gene;

(b) selecting an embryonic stem cell from step (a) wherein the DNA sequence encoding a selectable marker has integrated into the genomic DNA of said embryonic cell;

(c) introducing the selected embryonic stem cell from step (b) into a blastocyst of a pseudopregnant mouse, and allowing the blastocyst to develop into a viable transgenic mouse homozygous for a disrupted VEGF-B gene and which results lacks of expression of VEGF-B.

7. The method of claim 6, wherein the disrupted VEGF-B gene comprises a polynucleotide sequence from a VEGF-B gene wherein at least one codon for cysteine has been changed or deleted.

8. The method as claimed in claim 6, wherein the 5′

region of homology to the genomic VEGF-B comprises exon 1 of the VEGF-B gene.

9. The method as claimed in claim 6, wherein the 3′

region of homology to the genomic VEGF-B comprises exon 7 of the VEGF-B gene.

12. The transgenic mouse of claim 1, wherein said DNA sequence comprises a 5′

region and a 3′

region of homology to the genomic VEGF-B gene.

13. A method for screening a compound for angiogenic or anti-angiogenic activity, said method comprising introducing the compound into a transgenic mouse of claim 1, and assaying the vascular development in the animal.

14. A method for screening a compound for tumor growth modulating activity in a transgenic mouse, said method comprising introducing a tumor cell into a transgenic mouse of claim 1, introducing the compound into the same mouse, and assaying tumor development in the mouse.

10. A mouse embryonic stem cell homozygous for a disrupted VEGF-B gene, wherein said disrupted VEGF-B gene comprises a polynucleotide sequence from a VEGF-B gene wherein at least one codon for cysteine has been changed or deleted.

11. A mouse embryonic stem cell homozygous for a disrupted VEGF-B gene, wherein said VEGF-B gene is disrupted by the insertion of a DNA sequence which comprises a 5′

region of homology to the genomic VEGF-B comprising exon 1, and a 3′

region of homology to the genonic VEGF-B comprising exon 7.