Method for genetically modifying isolated non-human mammalian cell by homologous recombination

• US 8,846,402 B2
• Filed: 12/19/2012
• Issued: 09/30/2014
• Est. Priority Date: 10/31/2000
• Status: Expired due to Fees

• Current Assignee: Regeneron Pharmaceuticals Incorporated
• Original Assignee: Regeneron Pharmaceuticals Incorporated
• Inventors: Economides, Aris N., Murphy, Andrew J., Valenzuela, David M., Frendewey, David, Yancopoulos, George D.
• Primary Examiner(s): Ton, Thaian N
• Assistant Examiner(s): Moloye, Titilayo
• Application Number:
  US13/719,804
• Publication Number:
  US 20130137101A1
• Time in Patent Office: 650 Days
• Field of Search: None
• US Class Current: 435/463
• 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: C12N 15/79 Vectors or expression systems specially adapted for eukaryotic hosts
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2810/10 Vectors comprising a non-peptidic targeting moiety
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism

Abstract

A method for engineering and utilizing large DNA vectors to target, via homologous recombination, and modify, in any desirable fashion, endogenous genes and chromosomal loci in eukaryotic cells. These large DNA targeting vectors for eukaryotic cells, termed LTVECs, are derived from fragments of cloned genomic DNA larger than those typically used by other approaches intended to perform homologous targeting in eukaryotic cells. Also provided is a rapid and convenient method of detecting eukaryotic cells in which the LTVEC has correctly targeted and modified the desired endogenous gene(s) or chromosomal locus (loci) as well as the use of these cells to generate organisms bearing the genetic modification.

Claims

1. A method for targeting a cloned genomic fragment to a desired position in an isolated non-human mammalian cell genome, comprising

(a) obtaining a vector comprising a donor fragment having a homology box 1 (hb1) of at least 40 nucleotides in length upstream and a homology box 2 (hb2) of at least 40 nucleotides in length downstream appended to a human genomic fragment containing the genomic sequence to be inserted into the genome of an isolated non-human mammalian cell;

(b) obtaining a second vector comprising a target fragment isolated from a non-human mammalian cell, wherein the second vector accommodates DNA fragments greater than 20 kb;

(c) introducing both vectors into a bacterium;

(d) permitting bacterial homologous recombination between the donor fragment and the target fragment such that the human genomic fragment is inserted into a region to be modified in the non-human mammalian genomic target fragment, thereby producing a Large Targeting Vector for use in Eukaryotic Cells (LTVEC) comprising a modified non-human mammalian genomic fragment;

(e) introducing the LTVEC into the isolated non-human mammalian cell to target and modify an endogenous chromosomal locus in the isolated non-human mammalian cell, thereby forming a modified locus comprising the human DNA sequence resulting in a modified non-human mammalian cell; and

,

(f) assaying the modified non-human mammalian cell for the modified locus.

2. The method of claim 1, wherein the isolated non-human mammalian cell is a mammalian embryonic stem cell.

3. The method of claim 2, wherein the mammalian embryonic stem cell is a rodent embryonic stem cell.

4. The method of claim 3, wherein the rodent embryonic stem cell is selected from a mouse embryonic stem cell and a rat embryonic stem cell.

5. The method of claim 1, wherein the human genomic fragment comprises a human gene or gene fragment.

6. The method of claim 1, wherein the human genomic fragment comprises a DNA sequence that is orthologous to a coding sequence of the endogenous chromosomal locus of the isolated non-human mammalian cell.

7. The method of claim 6, wherein the coding sequence of the endogenous chromosomal locus of the isolated non-human mammalian cell is replaced with the orthologous DNA sequence.

8. The method of claim 1, wherein the method results in an insertion of a new coding region, an insertion or replacement of a gene segment, or an insertion or replacement of a regulatory element in the endogenous chromosomal locus of the isolated non-human mammalian cell.

9. The method of claim 1, wherein the method results in a replacement of the endogenous chromosomal locus of the isolated non-human mammalian cell.

10. The method of claim 1, wherein the method results in the creation of a conditional allele in the endogenous chromosomal locus of the isolated non-human mammalian cell.

11. The method of claim 1, wherein the second vector accommodates DNA fragments greater than 100 kb.

12. The method of claim 1, wherein the LTVEC is greater than 140 kb.

13. The method of claim 1, wherein the LTVEC is greater than 190 kb.

14. The method of claim 1, wherein the LTVEC is greater than 235 kb.

15. The method of claim 1, wherein the LTVEC is greater than 256 kb.

16. The method of claim 1, wherein the LTVEC is greater than 265 kb.

17. The method of claim 1, wherein the LTVEC is greater than 288 kb.

18. The method of claim 1, wherein the LTVEC is greater than 308 kb.

19. The method of claim 1, wherein the LTVEC is greater than 322 kb.

20. The method of claim 1, wherein the LTVEC is greater than 368 kb.

21. The method of claim 1, wherein assaying the cell for the modified locus comprises detecting modification of a parental allele.

22. The method of claim 1, wherein the LTVEC comprises a sequence selected from a loxP site and a FRT site.

23. A method for targeting a cloned genomic fragment to a desired position in an isolated non-human mammalian cell genome, comprising (a) obtaining a vector comprising a donor fragment having a homology box 1 (hb1) of at least 40 nucleotides in length upstream and a homology box 2 (hb2) of at least 40 nucleotides in length downstream appended to a human genomic fragment containing the genomic sequence to be inserted into the genome of an isolated non-human mammalian cell and orthologous to an endogenous chromosomal locus in the isolated non-human mammalian cell;

(b) obtaining a second vector comprising a target fragment isolated from a non-human mammalian cell, wherein the second vector accommodates DNA fragments greater than 20 kb;

(c) introducing both vectors into a bacterium;

(d) permitting bacterial homologous recombination between the donor fragment and the target fragment such that the human genomic fragment is inserted into a region to be modified in the non-human mammalian genomic target fragment, thereby producing a Large Targeting Vector for use in Eukaryotic Cells (LTVEC) comprising a modified non-human mammalian genomic fragment;

(e) introducing the LTVEC into the isolated non-human mammalian cell to target and modify the endogenous chromosomal locus in the isolated non-human mammalian cell, thereby forming a modified locus comprising the human DNA sequence resulting in a modified non-human mammalian cell; and

,

(f) assaying the modified non-human mammalian cell for the modified locus.

24. The method of claim 23, wherein the isolated non-human mammalian cell is a mammalian embryonic stem cell.

25. The method of claim 24, wherein the mammalian embryonic stem cell is a rodent embryonic stem cell.

26. The method of claim 25, wherein the rodent embryonic stem cell is selected from a mouse embryonic stem cell and a rat embryonic stem cell.

27. The method of claim 23, wherein the human genomic fragment comprises a human gene or gene fragment.

28. The method of claim 23, wherein the orthologous human genomic sequence is a gene coding sequence.

29. The method of claim 26, wherein the method results in the replacement of a murine genetic locus with an orthologous human genetic locus.

30. The method of claim 23, wherein the method results in an insertion of a new coding region, an insertion or replacement of a gene segment, or an insertion or replacement of a regulatory element in the endogenous chromosomal locus of the isolated non-human mammalian cell.

31. The method of claim 23, wherein the method results in a replacement of the endogenous chromosomal locus of the isolated non-human mammalian cell.

32. The method of claim 23, wherein the method results in the creation of a conditional allele in the endogenous chromosomal locus of the isolated non-human mammalian cell.

33. The method of claim 23, wherein the second vector accommodates DNA fragments greater than 100 kb.

34. The method of claim 23, wherein the LTVEC is greater than 140 kb.

35. The method of claim 23, wherein the LTVEC is greater than 190 kb.

36. The method of claim 23, wherein the LTVEC is greater than 235 kb.

37. The method of claim 23, wherein the LTVEC is greater than 256 kb.

38. The method of claim 23, wherein the LTVEC is greater than 265 kb.

39. The method of claim 23, wherein the LTVEC is greater than 288 kb.

40. The method of claim 23, wherein the LTVEC is greater than 308 kb.

41. The method of claim 23, wherein the LTVEC is greater than 322 kb.

42. The method of claim 23, wherein the LTVEC is greater than 368 kb.

43. The method of claim 23, wherein assaying the cell for the modified locus comprises detecting modification of a parental allele.

44. The method of claim 23, wherein the LTVEC comprises a sequence selected from a loxP site and a FRT site.

45. A method for modifying a desired position in an isolated non-human mammalian cell genome, comprising

(a) obtaining a vector comprising a donor fragment having a homology box 1 (hb1) of at least 40 nucleotides in length upstream and a homology box 2 (hb2) of at least 40 nucleotides in length downstream appended to a DNA sequence to be inserted into the genome of an isolated non-human mammalian cell;

(b) obtaining a second vector comprising a target fragment isolated from a non-human mammalian cell, wherein the second vector accommodates DNA fragments greater than 20 kb;

(c) introducing both vectors into a bacterium;

(d) permitting bacterial homologous recombination between the donor fragment and the target fragment such that the DNA sequence is inserted into a region to be modified in the non-human mammalian genomic target fragment, thereby producing a Large Targeting Vector for use in Eukaryotic Cells (LTVEC) comprising a modified non-human mammalian genomic fragment;

(e) introducing the LTVEC into the isolated non-human mammalian cell to target and modify an endogenous chromosomal locus in the isolated non-human mammalian cell thereby forming a modified locus comprising the DNA sequence resulting in a modified non-human mammalian cell; and

,

(f) assaying the modified non-human mammalian cell for the modified locus.

46. The method of claim 45, wherein the modification of the isolated non-human mammalian genome comprises a deletion of a coding sequence, gene segment or regulatory element.

47. The method of claim 45, wherein the modification of the isolated non-human mammalian genome comprises an alteration of a coding sequence, gene segment or regulatory element.

48. The method of claim 47, wherein the modification of the isolated non-human mammalian genome comprises substitution of an endogenous gene or locus with an orthologous gene or locus.

49. The method of claim 45, wherein the modification of the isolated non-human mammalian genome comprises an insertion of a coding region, gene segment or regulatory element.

50. The method of claim 49, wherein the modification of the isolated non-human mammalian genome comprises an insertion of a reporter gene into an endogenous gene, wherein the reporter gene is under transcriptional control of the endogenous gene.

51. The method of claim 50, wherein the reporter gene is β

-galactosidase (LacZ).

52. The method of claim 45, wherein the modification of the isolated non-human mammalian genome comprises the creation of a conditional allele.

53. The method of claim 45, wherein the modification of the isolated non-human mammalian genome comprises the introduction of loxP and/or FRT sites.

54. The method of claim 45, wherein the modification of the isolated non-human mammalian genome comprises the replacement of an endogenous coding sequence or gene segment with an orthologous coding sequence or gene segment.