Procedure for specific replacement of a copy of a gene present in the recipient genome by the integration of a gene different from that where the integration is made

• US 6,528,314 B1
• Filed: 06/06/1995
• Issued: 03/04/2003
• Est. Priority Date: 03/20/1989
• Status: Expired due to Term

• Current Assignee: Institut Pasteur
• Original Assignee: Institut Pasteur
• Inventors: Brulet, Philippe, Le Mouellic, Hervé
• Primary Examiner(s): Brusca, John S.
• Application Number:
  US08/466,539
• Time in Patent Office: 2,828 Days
• Field of Search: 435/172.3, 435/461, 435/463, 536/23.1, 536/23.5, 536/23.7, 536/23.72
• US Class Current: 435/461
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2800/108 episomal vectors
• 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 2830/00 Vector systems having a special element relevant for transcription
• CPC Class Codes: C12N 2830/85 mammalian

Abstract

A process for providing a recombinant, heterologous gene in the genome of a eukaryotic cell is provided. In particular, heterologous DNA is inserted into a recipient gene of the eukaryotic cell by homologous recombination. Moreover, a transgenic or chimeric animal comprising cells with DNA inserted into its genome by homologous recombination is disclosed. Further, the method of making these transgenic animal is taught.

Claims

1. An in vitro process for providing a recombinant, heterologous gene in a genome of a mammalian cell, wherein the process comprises:

(A) providing a mammalian cell having a recipient gene in its genome, wherein said recipient gene comprises complementing DNA comprising a first nucleotide sequence and a second nucleotide sequence downstream of said first nucleotide sequence;

(B) transfecting said mammalian cell in vitro with a DNA comprising;

(1) a third nucleotide sequence homologous to said first nucleotide sequence;

(2) a fourth nucleotide sequence homologous to said second nucleotide sequence; and

(3) a DNA sequence heterologous with respect to DNA in said recipient gene, wherein said heterologous DNA sequence is between said third and said fourth nucleotide sequences and said heterologous DNA sequence comprises a first insertion DNA sequence and a second insertion DNA sequence, wherein said first insertion DNA sequence comprises a first coding sequence that encodes a first product that does not confer resistance to a selection agent involved in the selection of transformants, and a regulatory sequence for regulating the expression of said first product, and said second insertion DNA sequence comprises a second coding sequence that encodes a second product that confers resistance to a selection agent involved in the selection of transformants, and a promoter allowing the expression of said second product in said mammalian cell; and

(C) selecting a transfected cell in which said heterologous DNA sequence has been inserted between said first and said second nucleotide sequences in said recipient gene by homologous recombination with said third and said fourth nucleotide sequences to thereby provide a mammalian cell containing in its genome the recombinant, heterologous gene, and in addition, the second insertion DNA sequence encoding the selective agent.

2. The in vitro process as claimed in claim 1, wherein said regulatory sequence is a promoter.

3. The in vitro process as claimed in claim 1, wherein said regulatory sequence is an enhancer.

4. The in vitro process as claimed in claim 1, wherein insertion of said heterologous DNA in said recipient gene is accompanied by replacement of recipient gene DNA.

5. The in vitro process as claimed in claim 1, wherein said recipient gene contains a promoter, which is inactivated following insertion of said heterologous DNA sequence into said recipient gene.

6. The in vitro process as claimed in claim 1, wherein said recipient gene has a regulatory sequence and said coding sequence of said first insertion DNA sequence is under the control of said regulatory sequence following insertion of said heterologous DNA sequence into said recipient gene.

7. The in vitro process as claimed in claim 1, wherein expression of said recipient gene is under the control of said regulatory sequence following insertion of said heterologous DNA sequence in said recipient gene.

8. The in vitro process as claimed in claim 1, wherein each of said third and said fourth nucleotide sequences has a length greater than 150 base pairs and shorter than the length of said recipient gene.

9. The in vitro process as claimed in claim 1, wherein said recipient gene is present in said mammalian cell in at least two copies and said heterologous DNA sequence is inserted into only one copy of said recipient gene.

10. The in vitro process as claimed in claim 1, wherein said first coding sequence encodes an interferon, an interleukin, a β

-3-adrenergic receptor, a retinoic acid receptor, an HIV receptor, or lac Z.

11. The in vitro process as claimed in claim 1, wherein said first coding sequence encodes lac Z.

12. The in vitro process as claimed in claim 1, wherein said mammalian cell is a mouse embryonic stem (E. S.) cell.

13. The in vitro process as claimed in claim 1, wherein said transfection is by electroporation.

14. The in vitro process as claimed in claim 1, which further comprises amplifying by polymerase chain reaction the heterologous DNA sequence at the locus at which the insertion is made.

15. The in vitro process as claimed in claim 1, wherein the selective agent is neoR.

16. The in vitro process as claimed in claim 1, wherein said recipient gene codes for a receptor for an infectious agent, said recipient gene is present in said mammalian cell in at least two copies, and said heterologous DNA sequence is inserted into only one copy of said recipient gene.

17. Mammalian cells transformed according to the in vitro process of claim 1.

18. Mammalian cells according to claim 17, wherein said mammalian cells comprise mouse embryonic stem (E. S.) cells.

19. The in vitro process as claimed in claim 1, wherein said recipient gene is not expressed in said mammalian cell and expression of said recipient gene is made possible following insertion of said heterologous DNA sequence in said recipient gene.

20. Mammalian cells transformed according to the in vitro process of claim 19.

21. Mammalian cells according to claim 20, wherein said mammalian cells comprise mouse embryonic stem (E. S.) cells.

22. The in vitro process as claimed in claim 1, wherein expression of said recipient gene is modified following insertion of said heterologous DNA sequence.

23. The in vitro process as claimed in claim 1, wherein said second insertion DNA sequence lacks a polyadenylation sequence and is operably linked at the 3′

end to a 5′

region on an intron containing a sequence that causes the selective degradation of transcripts of said heterologous DNA sequence.

24. The in vitro process as claimed in claim 1, wherein the process further comprises achieving a homologous recombination frequency ratio of at least 1 homologous recombination event per 900 random insertion events.

25. The in vitro process as claimed in claim 23, wherein the process further comprises achieving a homologous recombination frequency ratio of at least 1 homologous recombination event per 40 random insertion events.

27. The in vitro process as claimed in claim 2, wherein said regulatory sequence is a promoter.

28. The in vitro process as claimed in claim 2, wherein said regulatory sequence is an enhancer.

30. The in vitro process as claimed in claim 2, wherein said recipient gene contains a promoter, which is inactivated following insertion of said heterologous DNA sequence into said recipient gene.

31. The in vitro process as claimed in claim 2, wherein expression of said recipient gene is under the control of said regulatory sequence following insertion of said heterologous DNA sequence in said recipient gene.

32. The in vitro process as claimed in claim 2, wherein each of said third and said fourth nucleotide sequences has a length greater than 150 base pairs and shorter than the length of said recipient gene.

33. The in vitro process as claimed in claim 2, wherein said recipient gene is present in said mammalian cell in at least two copies and said heterologous DNA sequence is inserted into only one copy of said recipient gene.

34. The in vitro process as claimed in claim 2, wherein said mammalian cell is a mouse embryonic stem (E. S.) cell.

35. The in vitro process as claimed in claim 2, wherein said transfection is by electroporation.

36. The in vitro process as claimed in claim 2, which further comprises amplifying by polymerase chain reaction the heterologous DNA sequence at the locus at which the insertion is made.

37. The in vitro process as claimed in claim 2, wherein the selective agent is neoR.

38. The in vitro process as claimed in claim 2, wherein said recipient gene codes for a receptor for an infectious agent, said recipient gene is present in said mammalian cell in at least two copies, and said heterologous DNA sequence is inserted into only one copy of said recipient gene.

41. The in vitro process as claimed in claim 2, wherein said recipient gene is not expressed in said eukaryotic cell and expression of said recipient gene is made possible following insertion of said heterologous DNA sequence in said recipient gene.

42. Mammalian cells transformed according to the in vitro process of claim 41.

43. Mammalian cells according to claim 42, wherein said mammalian cells comprise mouse embryonic stem (E. S.) cells.

48. A recombinant heterologous gene made according to the in vitro process of claim 1.

26. An in vitro process for providing a recombinant, heterologous gene in a genome of a mammalian cell, wherein the process comprises:

(A) providing a mammalian cell having a recipient gene in its genome, wherein said recipient gene comprises complementing DNA comprising a first nucleotide sequence and a second nucleotide sequence downstream of said first nucleotide sequence;

(B) transfecting said mammalian cell in vitro with a DNA comprising;

(1) a third nucleotide sequence homologous to said first nucleotide sequence;

(2) a fourth nucleotide sequence homologous to said second nucleotide sequence; and

(3) a DNA sequence heterologous with respect to DNA in said recipient gene, wherein said heterologous DNA sequence is between said third and said fourth nucleotide sequences and said heterologous DNA sequence comprises a first insertion DNA sequence and a second insertion DNA sequence, wherein said first insertion DNA sequence comprises a regulatory sequence, and said second insertion DNA sequence comprises a coding sequence that encodes a product that confers resistance to a selection agent involved in the selection of transformants, and a promoter allowing the expression of said product in said mammalian cell; and

(C) selecting a transfected cell in which said heterologous DNA sequence has been inserted between said first and said second nucleotide sequences in said recipient gene by homologous recombination with said third and said fourth nucleotide sequences to thereby provide a mammalian cell containing in its genome the recombinant, heterologous gene, and in addition, the second insertion DNA sequence encoding the selective agent.

29. The in vitro process as claimed in claim 26, wherein insertion of said heterologous DNA in said recipient gene is accompanied by replacement of recipient gene DNA.

39. Mammalian cells transformed according to the in vitro process of claim 26.

40. Mammalian cells according to claim 39, wherein said mammalian cells comprise mouse embryonic stem (E. S.) cells.

44. The in vitro process as claimed in claim 26, wherein expression of said recipient gene is modified following insertion of said heterologous DNA sequence.

45. The in vitro process as claimed in claim 26, wherein said second insertion DNA sequence lacks a polyadenylation sequence and is operably linked at the 3′

end to a 5′

region on an intron containing a sequence that causes the selective degradation of transcripts of said heterologous DNA sequence.

46. The in vitro process as claimed in claim 26, wherein the process further comprises achieving a homologous recombination frequency ratio of at least 1 homologous recombination event per 900 random insertion events.

47. The in vitro process as claimed in claim 45, wherein the process further comprises achieving a homologous recombination frequency ratio of at least 1 homologous recombination event per 40 random insertion events.

49. A recombinant heterologous gene made according to the in vitro process of claim 26.