Methods of constructing a gene mutation library and compounds and compositions thereof

• US 20070196917A1
• Filed: 10/12/2006
• Published: 08/23/2007
• Est. Priority Date: 03/13/1997
• Status: Abandoned Application

• Current Assignee: Geoffrey Hicks, Henry Ruley
• Original Assignee: Geoffrey Hicks, Henry Ruley
• Inventors: Hicks, Geoffrey, Ruley, Henry
• Application Number:
  US11/546,881
• Publication Number:
  US 20070196917A1
• Time in Patent Office: Days
• US Class Current: 435/353
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2217/072 maintaining or altering function, i.e. knock in
• 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 2267/0393 Animal model comprising a reporter system for screening tests
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic

Abstract

The invention is directed toward a method of producing a selected cell line or a non-human transgenic animal model for the analysis of the function of a gene comprising introducing into an embryonic stem cell a vector having a selectable marker which, when the vector is inserted within a gene, the inserted vector can inhibit the expression of the gene, selecting embryonic stem cells expressing the selectable marker, excising the vector from the embryonic stem cells expressing the selectable marker such that host DNA from the gene is linked to the excised vector, sequencing the host DNA in the excised vector, comparing the sequence of the host DNA to known gene sequences to determine which host DNA is from a gene for which a model for the analysis of the function the gene is desired, selecting the embryonic stem cell containing the inhibited gene for which a model for the analysis of gene function is desired, and forming a cell line or a non-human transgenic animal from the selected embryonic stem cell. The invention is also directed toward a method of selecting a cell for the analysis of the function of a gene. The invention is also directed toward libraries of cells, cell lines, and transgenic animals produced using cells produced by the methods disclosed herein.

Claims

1. A nucleic acid molecule comprising a cassette containing a splice acceptor site, a selectable marker gene, and an origin of replication, wherein said selectable marker gene and said origin of replication are downstream of said splice acceptor site, and wherein said cassette is flanked by a portion of a eukaryotic gene at its 3′

end, 5′

end, or both.

5. The nucleic acid molecule of claim 1, wherein said eukaryotic gene is a mammalian gene.

6. The nucleic acid molecule of claim 1, wherein said portion of said eukaryotic gene is a non-coding region of said gene.

7. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule is a transposon or a viral vector.

8. The nucleic acid molecule of claim 7, wherein said viral vector is a retroviral vector.

9. The nucleic acid molecule of claim 8, wherein said nucleic acid molecule further comprises an enhancement sequence that can be used to enhance the isolation of the retroviral vector.

10. The nucleic acid molecule of claim 9, wherein the enhancement sequence comprises the lac operator.

11. The nucleic acid molecule of claim 1, wherein said selectable marker gene is a host selectable marker gene.

12. The nucleic acid molecule of claim 1, wherein said selectable marker gene encodes a drug resistance gene, a luminescent gene, or chloramphenical acetyl transferase (CAT).

13. The nucleic acid molecule of claim 12, wherein said drug resistance gene is neomycin, hph gene, or Ecogpt gene.

14. The nucleic acid molecule of claim 12, wherein said luminescent gene encodes luciferase or a green fluorescent gene.

15. The nucleic acid molecule of claim 1, further comprising a second selectable marker gene.

16. The nucleic acid molecule of claim 15, wherein said second selectable marker is a prokaryotic selectable marker gene.

17. The nucleic acid molecule of claim 1, wherein said origin of replication is non-mammalian.

18. The nucleic acid molecule of claim 17, wherein said origin of replication is yeast or bacterial.

19. The nucleic acid molecule of claim 1, further comprising a termination sequence.

24. A cell comprising a nucleic acid molecule of claim 1, wherein said nucleic acid molecule is integrated into a host cell gene such that the expression of said selectable marker is under the control of regulatory elements of said host cell gene and wherein said origin of replication is exogenous to said host cell gene.

25. The cell of claim 24, wherein the expression of said host cell gene in said cell is decreased or inhibited as a result of insertion of said exogenous nucleic acid molecule in said host cell gene relative to such expression in a control cell.

26. The cell of claim 24, wherein said cell is an embryonic cell, an embryonic stem cell, or differentiated cell.

27. The cell of claim 24, wherein said cell is a murine or human cell.

28. A library of at least ten cells, each of which comprises an exogenous nucleic acid molecule of claim 1, wherein said nucleic acid molecule is integrated into a host cell gene such that the expression of said selectable marker is under the control of regulatory elements of said host cell gene and said origin of replication is exogenous to said cellular gene.

29. The library of claim 28, wherein expression of said host cell gene in at least one of said cells is decreased or inhibited as a result of insertion of said exogenous nucleic acid molecule in said host cell gene relative to such expression in a control cell.

2. A nucleic acid molecule comprising a cassette containing a selectable marker gene and an origin of replication 3′

to said selectable marker gene, such that the expression of said selectable marker gene is under the control of the promoter of a gene of a host cell upon the integration of said nucleic acid molecule in said host cell gene when said nucleic acid molecule is contacted with said host cell and wherein said origin of replication is exogenous to said host cell gene, and wherein said cassette is flanked by a portion of a eukaryotic gene at its 3′

end, 5′

end, or both.

3. A nucleic acid molecule that encodes an mRNA transcript containing an origin of replication and a selectable marker gene.

4. The nucleic acid molecule of claim 3, wherein said nucleic acid molecule comprises a cassette containing said origin of replication and said selectable marker gene and wherein said cassette is flanked at its 3′

end, 5′

end, or both by a portion of a eukaryotic gene.

20. A library of nucleic acid molecules, each of which comprises a cassette containing a splice acceptor site, a selectable marker gene, and an origin of replication wherein said selectable marker gene and said origin of replication, are downstream of said splice acceptor site, and wherein in at least one nucleic acid molecule of said library, said cassette is flanked by a portion of a eukaryotic gene at its 3′

end, 5′

end, or both.

21. A library of nucleic acid molecules, each of which comprises a cassette containing a selectable marker gene and an origin of replication 3′

to said selectable marker gene, such that the expression of said selectable marker gene is under the control of the promoter of a host cellular gene upon the integration of said nucleic acid molecule in said host cell gene when said nucleic acid molecule is contacted with a host cell and wherein said origin of replication is exogenous to said host cell gene, and wherein in at least one nucleic acid molecule of said library, said cassette is flanked by a portion of a eukaryotic gene at its 3′

end, 5′

end, or both.

22. A library of nucleic acid molecules, each of which encodes an mRNA transcript containing an origin of replication and a selectable marker.

23. The library of claim 22, wherein at least one nucleic acid molecule of said library comprises a cassette containing an origin of replication and a selectable marker, wherein said cassette is flanked at 3′

end, 5′

end, or both by a portion of a eukaryotic gene.