Tissue specific promoters and transgenic mouse for the screening of pharmaceuticals

• US 6,313,373 B1
• Filed: 10/29/1999
• Issued: 11/06/2001
• Est. Priority Date: 10/30/1998
• Status: Expired due to Fees

• Current Assignee: Case Western Reserve University
• Original Assignee: Case Western Reserve University
• Inventors: Crish, James F., Eckert, Richard L.
• Primary Examiner(s): Clark, Deborah J. R.
• Assistant Examiner(s): DRABIK, CHRISTOPHER E
• Application Number:
  US09/430,201
• Time in Patent Office: 739 Days
• Field of Search: 536/23.1, 435/320.1, 435/325, 435/69.1, 800/3, 800/8, 800/21
• US Class Current: 800/18
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/0306 Animal model for genetic diseases
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: C07K 14/82 Translation products from oncogenes
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 2830/008 cell type or tissue specific enhancer/promoter combination

Abstract

The present invention provides human involucrin (hINV) sequences having tissue specific and cell type specific promoter activity. The sequences provided herein direct expression to suprabasal cells of stratifying epithelia. The invention further provides methods for the production of transgenic animals which contain a hINV promoter sequence which directs the expression of human papillomavirus 16 oncogenes (or other oncogenes). These animals display cervical and epidermal hyperplasias as well as cancer of the trachea, esophagus, colon, epidermis, anus/rectum, lymph nodes, spleen and lung. The animals of the invention provide a useful model for screening potential anti-neoplastic compounds, carcinogens, and co-carcinogens for a number of cancers.

Claims

1. A purified oligonucleotide comprising the nucleotide sequence from nucleotide 1 to nucleotide 521 of SEO ID NO:

1.

2. The oligonucleotide of claim 1, wherein said oligonucleotide additionally comprises the operably linked nucleotide sequence from nucleotide 2434 to nucleotide 2467 of SEQ ID NO:

1.

3. The oligonucleotide of claim 1 or 2, wherein said nucleotide sequence is operably linked to a nucleic acid sequence of interest.

4. A recombinant expression vector comprising the nucleotide sequence from nucleotide 1 to nucleotide 521 of SEQ ID NO:

1.

5. The recombinant expression vector of claim 4, wherein said nucleotide sequence is operably linked to a nucleic acid sequence of interest.

6. A recombinant expression vector comprising the nucleotide sequence from nucleotide 1 to nucleotide 521 of SEQ ID NO:

1 operably linked to the sequence from nucleotide 2434 to nucleotide 2467 of SEQ ID NO;

1.

7. The recombinant expression vector of claim 6, wherein said nucleotide sequence is operably linked to a nucleic acid sequence of interest.

8. An isolated host cell comprising a recombinant expression vector wherein said recombinant expression vector comprises the nucleotide sequence from nucleotide 1 to nucleotide 521 of SEQ ID NO:

1.

9. A mouse cell comprising a recombinant expression vector wherein said recombinant vector comprises the nucleotide sequence from nucleotide 1 to nucleotide 521 of SEQ ID NO:

1.

10. The mouse cell of claim 9, wherein said mouse cell is a fertilized egg cell.

11. The mouse cell of claim 9, wherein said mouse cell is in a blastomere.

12. The mouse cell of claim 9, wherein said mouse cell is in an eight-cell embryo.

13. The mouse cell of claim 9, wherein said mouse cell is in a midgestation embryo.

14. The mouse cell of claim 9, wherein said mouse cell is an embryonic stem cell.

15. A transgenic mouse displaying tissue specific expression of a nucleic acid sequence of interest, wherein said transgenic mouse comprises the nucleotide sequence from nucleotide 1 to nucleotide 521 of SEQ ID NO:

1.

16. The transgenic mouse of claim 15, wherein said tissue is selected from the group consisting of uroepithelial tissue and stratified squamous epithelial tissue.

17. The transgenic mouse of claim 16, wherein said stratified squamous epithelial tissue is in an organ selected from the group consisting of epidermis, esophagus and cervix.

18. The transgenic mouse of claim 17, wherein said tissue specific expression is cell type specific.

19. The transgenic mouse of claim 18, wherein said cell in said stratified squamous epithelial tissue is suprabasal.

20. The transgenic mouse of claim 15, wherein said nucleic acid sequence of interest is a coding sequence of an oncogene.

21. The transgenic mouse of claim 20, wherein said oncogene is a human papillomavirus 16 oncogene.

28. A method of screening anti-neoplastic compounds, comprising:

a.) providing;

i) the transgenic mouse of claim 19 or 20; and

ii) a compound suspected of having anti-neoplastic activity;

b) administering said compound to said transgenic mouse to produce a treated transgenic mouse; and

c) detecting anti-neoplastic activity in said treated transgenic mouse, thereby identifying said compound as anti-neoplastic.

22. A method for selective expression of a nucleic acid sequence of interest in epithelial cells of a mouse, comprising:

a) providing;

i) a transgene, wherein said transgene contains the nucleotide sequence from nucleotide 1 to nucleotide 521 of SEQ ID NO;

1 operably linked to said nucleic acid sequence of interest;

ii) an embryonic stem cell of a mouse; and

iii) a pseudopregnant mouse; and

b) introducing;

said transgene into said embryonic stem cell to produce a transgenic embryonic stem cell; and

c) introducing;

said transgenic embryonic stem cell into said pseudopregnant mouse under conditions such that said pseudopregnant mouse delivers progeny of said transgenic embryonic stem cell, wherein said nucleic acid sequence of interest is selectively expressed in said epithelial cells of said progeny.

23. The method of claim 22, further comprising d) identifying at least one offspring of said progeny wherein said nucleic acid sequence of interest is selectively expressed in said epithelial cells of said offspring.

24. The method of claim 22, wherein said transgene further comprises the nucleotide sequence from nucleotide 2434 to nucleotide 2467 of SEQ ID NO:

1.

25. A method for producing a transgenic mouse, comprising:

a) providing;

i) a transgene, wherein said transgene contains the nucleotide sequence from nucleotide 1 to nucleotide 521 of SEQ ID NO;

1 operably linked to one or more oncogenes;

ii) an embryonic stem cell of a mouse; and

iii) a pseudopregnant mouse;

b) introducing;

said transgene into said embryonic stem cell to produce a transgenic embryonic stem cell; and

c) introducing;

said transgenic embryonic stem cell in to said pseudopregnant mouse under conditions such that said pseudopregnant mouse delivers progeny of said transgenic embryonic stem cell; and

d) identifying at least one offspring of said progeny, wherein

said oncogene is selectively expressed in epithelial cells of said offspring.

26. The method of claim 25, wherein said epithelial cell is suprabasal.

27. The method of claim 25, wherein said oncogene is selected from human papillomavirus 16 oncogne E6 nucleic acid sequence and human papilloma virus 16 oncogne E7 nucleic acid sequence.