Methods for increasing efficiency of homologous recombination

• US 20070155014A1
• Filed: 07/20/2006
• Published: 07/05/2007
• Est. Priority Date: 07/20/2005
• Status: Abandoned Application

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated), Regents of the University of California (University of California)
• Original Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated), Regents of the University of California (University of California)
• Inventors: Madden, Knut, Murray, James, Maga, Elizabeth, Bunting, Michaeline, Bertolini, Luciana
• Application Number:
  US11/490,474
• Publication Number:
  US 20070155014A1
• Time in Patent Office: Days
• US Class Current: 435/455
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 15/902 using homologous recombination
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/14 interfering N.A.

Abstract

Methods and compositions for increasing or decreasing homologous recombination activity in a eukaryotic cell are provided. More particularly, various methods and compositions are provided which decrease the level of non-homologous recombination in a cell and thereby increase the frequency of targeted homologous recombination events. Various compositions including cells and kits that can be employed in the methods are also provided.

Claims

1. A method for increasing the efficiency of homologous recombination of a polynucleotide of interest in a eukaryotic cell comprising

a) providing the eukaryotic cell having a decreased level of non-homologous recombination activity, wherein the decreased level of non-homologous recombination activity is transient;

b) introducing into said eukaryotic cell a composition comprising a homologous recombination cassette comprising a polynucleotide of interest and at least a first region having sufficient sequence identity to a corresponding first region of a target site in said eukaryotic cell, wherein said polynucleotide of interest is inserted into the target site by a homologous recombination event.

2. The method of claim 1, wherein said homologous recombination cassette comprises the polynucleotide of interest flanked by the first region and a second region having sufficient identity to a corresponding second region of the target site.

3. The method of claim 2, wherein said eukaryotic cell of step (a) comprises at least one polynucleotide comprising a silencing element, wherein said silencing element reduces the level of a gene product that contributes to non-homologous recombination.

4. The method of claim 3, wherein the gene product that contributes to non-homologous recombination encodes a Ku70 polypeptide or a Xrcc4 polypeptide.

5. The method of claim 3, wherein said silencing element comprises an siRNA silencing element, an miRNA silencing element, a double stranded RNA silencing element, a hairpin RNA silencing element, an antisense silencing element or a sense silencing element.

6. The method of claim 1, wherein said eukaryotic cell of step (a) further comprises at least one heterologous polynucleotide which when expressed in said eukaryotic cell increases homologous recombination activity in said eukaryotic cell.

7. The method of claim 1, wherein said composition of step (b) further comprises a polypeptide which increases homologous recombination activity in said eukaryotic cell.

8. A method for increasing the efficiency of homologous recombination of a polynucleotide of interest in a eukaryotic cell comprising

a) introducing into said eukaryotic cell a first polypeptide or a first polynucleotide which transiently decreases the level of non-homologous recombination in said eukaryotic cell;

and,

b) introducing into said eukaryotic cell a composition comprising a homologous recombination cassette comprising a polynucleotide of interest and a first region having sufficient sequence identity to a corresponding first region of a target site in said eukaryotic cell, wherein said polynucleotide of interest is integrated into the target site by a homologous recombination event.

9. The method of claim 8, wherein said homologous recombination cassette comprises the polynucleotide of interest flanked by the first region and a second region having sufficient identity to a corresponding second region of the target site.

10. The method of claim 9, wherein said first polynucleotide of step (a) comprises a silencing element, said silencing element reduces the level of a gene product that contributes to non-homologous recombination.

11. The method of claim 10, wherein the gene product that contributes to non-homologous recombination encodes a Ku70 polypeptide or a Xrcc4 polypeptide.

12. The method of claim 9, wherein said polynucleotide encoding said silencing element is stably incorporated into the eukaryotic cell and is operably linked to an inducible promoter.

13. The method of claim 10, wherein said silencing element comprises an siRNA silencing element, an miRNA silencing element, a double stranded RNA silencing element, a hairpin RNA silencing element, an antisense silencing element or a sense silencing element.

14. The method of claim 8, wherein said target site is endogenous to said eukaryotic cell or said target site is heterologous to said eukaryotic cell.

15. The method of claim 8, wherein said target site is chromosomally located or extrachromosomally located in said eukaryotic cell.

16. The method of claim 8, wherein said eukaryotic cell is a mammalian cell.

17. The method of claim 8, wherein said method further comprises selecting said eukaryotic cell having said polynucleotide of interest integrated into the target site.

18. The method of claim 8, wherein said method further comprises introducing into said eukaryotic cell at least a second polynucleotide which when expressed in said eukaryotic cell increases homologous recombination activity in said eukaryotic cell.

19. The method of claim 18, wherein said second polynucleotide encodes a RecA polypeptide, a Rad54 polypeptide, or a Rad51 polypeptide.

20. The method of claim 8, wherein said composition of step (b) further comprises a polypeptide which increases homologous recombination activity in said eukaryotic cell.

21. The method of claim 20, wherein said polypeptide which increases homologous recombination activity in said eukaryotic cell is selected from the group consisting of a RecA polypeptide, a Rad54 polypeptide, or a Rad51 polypeptide.

22. A kit comprising a polynucleotide encoding a silencing element, wherein said silencing element when introduced into a eukaryotic cell reduces the level of a gene product that contributes to non-homologous recombination, and, increases the homologous recombination activity in said eukaryotic cell;

said kit further comprises one or more polynucleotides selected from the group consisting of;

a) a polynucleotide comprising a homologous recombination cassette comprising at least a first region having sufficient sequence identity to a corresponding first region of a target site in said eukaryotic cell;

or

b) a polynucleotide which when expressed in said eukaryotic cell increases homologous recombination activity in said eukaryotic cell.

23. The kit of claim 22, wherein said homologous recombination cassette comprises the first region and a second region having sufficient identity to a corresponding second region of the target site.

24. The kit of claim 22, wherein the gene product that contributes to non-homologous recombination encodes a Ku70 polypeptide or a Xrcc4 polypeptide.

25. The kit of claim 22, wherein said silencing element encoded by said polynucleotide comprises an siRNA silencing element, an miRNA, a double stranded RNA silencing element, a hairpin RNA silencing element, an antisense silencing element or a sense silencing element.

26. The kit of claim 22, wherein said polynucleotide which when expressed in said eukaryotic cell increases homologous recombination activity is selected from the group consisting of a polynucleotide encoding a RecA polypeptide, a Rad54 polypeptide, or a Rad51 polypeptide.

27. A kit comprising a eukaryotic cell having a decreased level of non-homologous recombination activity, wherein the decreased level of non-homologous recombination activity is transient, said kit further comprises one or more polynucleotides selected from the group consisting of:

a) a polynucleotide comprising a homologous recombination cassette;

or

b) a polynucleotide which when expressed in said eukaryotic cell increases homologous recombination activity in said eukaryotic cell.

28. The kit of claim 27, wherein said eukaryotic cell comprises at least one heterologous polynucleotide stably incorporated into the cell comprising a silencing element operably linked to an inducible promoter, said silencing element reduces the level of a gene product that contributes to non-homologous recombination.

29. The kit of claim 28, wherein the gene product that contributes to non-homologous recombination encodes a Ku70 polypeptide or a Xrcc4 polypeptide.

30. The kit of claim 28, wherein said silencing element encoded by said polynucleotide comprises a siRNA silencing element, a miRNA, a double stranded RNA silencing element, a hairpin RNA silencing element, an antisense silencing element or a sense silencing element.

31. The kit of claim 27, wherein said polynucleotide which when expressed in said eukaryotic cell increases homologous recombination activity is selected from the group consisting of a polynucleotide encoding a RecA polypeptide, a Rad54 polypeptide, or a Rad51 polypeptide.

32. A method for increasing the efficiency of homologous recombination with an exogenous nucleic acid in a eukaryotic cell, the method comprising genetically modifying the cell with an expression construct that comprises a nucleotide sequence encoding an siRNA that specifically reduces the level of a gene product that contributes to non-homologous recombination, wherein the efficiency of homologous recombination is increased.