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

• US 7,371,577 B2
• Filed: 07/18/2005
• Issued: 05/13/2008
• Est. Priority Date: 03/08/1991
• Status: Expired due to Fees

• Current Assignee: Salk Institute for Biological Studies
• Original Assignee: Salk Institute for Biological Studies
• Inventors: Wahl, Geoffrey M., O'Gorman, Stephen V.
• Primary Examiner(s): BERTOGLIO, VALARIE E
• Application Number:
  US11/184,156
• Publication Number:
  US 20050257276A1
• Time in Patent Office: 1,030 Days
• Field of Search: None
• US Class Current: 435/462
• 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 vertebrate development. The FLP recombination system of the present invention can be incorporated in transgenic, non-human mammals to achieve site-specific integration of transgenes, to construct functional genes or to disrupt existing genes.

Claims

1. A co-transfection assay for the occurrence of FLP-mediated recombination, said assay comprising:

(a) co-transfecting a host mammalian cell with;

(i) a FLP expression plasmid comprising a promoter operably linked to a gene encoding FLP recombinase wherein said promoter is active in said cell, and

(ii) a reporter plasmid comprising a nonfunctional reporter gene wherein said nonfunctional reporter gene is inactivated by the presence of extraneous DNA flanked by FLP recombination target sites; and

(b) monitoring said host cell under a variety of conditions for the gain of expression of functional reporter gene product.

3. A co-transfection assay according to claim 1 wherein said FLP recombinase has the sequence set forth in SEQ ID NO:

2.

4. A co-transfection assay according to claim 1 wherein said FLP recombination target sites have the sequence as set forth in SEQ ID NO:

3.

5. A co-transfection assay according to claim 1 wherein said promoter is Pcmv, the cytomegalovirus immediate early promoter.

6. A co-transfection assay according to claim 1 wherein said reporter gene is operably linked to Psv, the early promoter from SV40.

2. A co-transfection assay for the occurrence of FLP-mediated recombination, said assay comprising:

(a) co-transfecting a host mammalian cell with;

(i) a FLP expression plasmid comprising a first promoter operably linked to a gene encoding FLP recombinase wherein said first promoter is active in said cell, and

(ii) a reporter plasmid comprising a functional reporter gene operably linked to a second promoter active in said cell wherein said reporter gene comprises at least one FLP recombination target site therein; and

(b) monitoring said host cell under a variety of conditions for the loss of expression of functional reporter gene product.

7. A co-transfection assay according to claim 2 wherein said FLP recombinase has the sequence set forth in SEQ ID NO:

2.

8. A co-transfection assay according to claim 2 wherein said FLP recombination target site has the sequence as set forth in SEQ ID NO:

3.

9. A co-transfection assay according to claim 2 wherein said first promoter operably linked to a gene encoding FLP recombinase is Pcmv, the cytomegalovirus early promoter.

10. A co-transfection assay according to claim 2 wherein said second promoter of the reporter plasmid is Psv, the early promoter from SV40.