FLP-mediated gene modification in mammalian cells, and compositions and cells useful therefor

• US 5,885,836 A
• Filed: 04/08/1997
• Issued: 03/23/1999
• Est. Priority Date: 03/08/1991
• Status: Expired due to Term

• Current Assignee: Salk Institute for Biological Studies
• Original Assignee: Salk Institute for Biological Studies
• Inventors: O'Gorman, Stephen V., Wahl, Geoffrey M.
• Primary Examiner(s): Low, Christopher S. F.
• Application Number:
  US08/825,784
• Time in Patent Office: 714 Days
• Field of Search: 435/325, 435/172.3, 435/320.1, 435/350, 435/351, 435/352, 435/353, 435/354, 435/363, 435/366, 435/193, 435/197, 435/194, 435/455, 435/462, 435/463, 435/466, 435/468 , 435/471, 536/24.2, 536/24.1, 536/23.74, 536/23.1, 935/66, 935/8, 935/22, 935/44, 800/2, 800/8, 800/3, 514/44
• US Class Current: 435/455
• 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: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/90 Stable introduction of foreign DNA into chromosome
• CPC Class Codes: C12N 15/907 in mammalian cells
• 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 9/00 Enzymes; Proenzymes; Compositions thereof preparations containing enzymes for cleaning teeth A61K8/66, A61Q11/00; medicinal preparations containing enzymes or proenzymes A61K38/43; enzyme containing detergent compositions C11D; enzymes with nucleic acid structure, e.g. ribozymes, C12N15/113; Processes for preparing, activating, inhibiting, separating or purifying enzymes preparation of malt C12C1/00

Abstract

A gene activation/inactivation and site-specific integration system has been developed for mammalian cells. The invention system is based on the recombination of transfected sequences by FLP, a recombinase derived from Saccharomyces. In several cell lines, FLP has been shown to rapidly and precisely recombine copies of its specific target sequence. For example, a chromosomally integrated, silent β-galactosidase reporter gene was activated for expression by FLP-mediated removal of intervening sequences to generate clones of marked cells. Alternatively, the reverse reaction can be used to target transfected DNA to specific chromosomal sites. These results demonstrate that FLP can be used, for example, to mosaically activate or inactivate transgenes for a variety of therapeutic purposes, as well as for analysis of vertebriate development.

Claims

1. A method for excising a nucleic acid sequence from the genome of a mammalian host cell, wherein said nucleic acid sequence is located in the genome of said mammalian host cell between a first FLP recombination target site (FRT) and a second FRT, said method comprising introducing into said mammalian host cell an FLP recombinase, wherein said FLP recombinase catalyzes recombination between said first FRT and said second FRT, thereby excising said nucleic acid sequence from said genome.

2. A method according to claim 1 wherein the excision of said sequence from said genome assembles a functional expression unit.

3. A method according to claim 2 wherein said functional expression unit is a marker gene.

4. A method according to claim 1 wherein the excision of said nucleic acid sequence from said genome disassembles a functional expression unit.

5. A method according to claim 4 wherein said functional expression unit is a marker gene.

6. A method according to claim 1 wherein said FLP recombinase is introduced into said mammalian host cell by injection of said FLP recombinase, microinjection of said FLP recombinase, or transfection with a FLP expression vector.

7. A method according to claim 6 wherein said FLP recombinase is introduced into said mammalian host cell by transfection with a FLP expression vector.

8. A method according to claim 7 wherein the expression of said FLP recombinase is regulated.

9. A method according to claim 1 wherein said FLP recombinase is obtained from a species of the genus Saccharomyces.

10. A method according to claim 1 wherein said FLP recombinase is obtained from a strain of Saccharomyces cerevisiae.

11. A method according to claim 1 wherein said FLP recombinase is encoded by the sequence set forth as SEQ ID NO:

1.

12. A method according to claim 1 wherein said nucleic acid sequence comprises an exogenous gene.

13. A method according to claim 1 wherein said nucleic acid sequence comprises at least one restriction endonuclease recognition site that is distinct from said first FRT and said second FRT.

14. A method according to claim 1 wherein said nucleic acid sequence comprises at least one marker gene.

15. A method according to claim 1 wherein said nucleic acid sequence comprises a bacterial origin of replication.

16. A method according to claim 1 wherein said nucleic acid sequence comprises a mammalian cellular or viral origin of DNA replication.

17. A method according to claim 1 wherein said nucleic acid sequence comprises:

a) an exogenous gene;

b) at least one restriction endonuclease recognition site that is distinct from said first FRT and said second FRT;

c) at least one marker gene;

d) a bacterial origin of replication; and

optionally

e) a mammalian cellular or viral origin of DNA replication.

18. A method for the recovery of an isolated nucleic acid sequence from the genome of a mammalian host cell, wherein said nucleic acid sequence is bordered on the 5'"'"' and 3'"'"' ends with a FRT, said method comprising:

a) contacting genomic DNA from said host cell with FLP recombinase, thereby excising said nucleic acid sequence from said genome, and then

b) recovering said excised nucleic acid sequence.

19. An isolated DNA construct comprising an insert segment flanked by a first FRT and a second FRT.

20. A DNA construct according to claim 19, wherein said insert segment comprises an exogenous gene.

21. A DNA construct according to claim 19, wherein said insert segment comprises at least one restriction endonuclease recognition site that is distinct from said first FRT and said second FRT.

22. A DNA construct according to claim 19, wherein said insert segment comprises at least one marker gene.

23. A DNA construct according to claim 19, wherein said insert segment comprises a bacterial origin of replication.

24. A DNA construct according to claim 19, wherein said insert segment comprises a mammalian cellular or viral origin of DNA replication.

25. A DNA construct according to claim 19, wherein said insert segment comprises:

1) an exogenous gene;

2) at least one restriction endonuclease recognition site that is distinct from said first FRT and said second FRT;

3) at leapt one marker gene;

4) a bacterial origin of replication; and

optionally

5) a mammalian cellular or viral origin of DNA replication.