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

• US 5,677,177 A
• Filed: 06/07/1995
• Issued: 10/14/1997
• 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: Wahl, Geoffrey M., O'Gorman, Stephen V.
• Primary Examiner(s): Low, Christopher S. F.
• Application Number:
  US08/486,409
• Time in Patent Office: 860 Days
• Field of Search: 800/2, 435/172.1, 435/172.2, 435/172.3, 435/240.2, 435/240.21, 435/320.1, 435/350, 435/351, 435/352, 435/353, 435/354, 435/363, 435/193, 435/197, 435/194, 514/44, 536/23.1, 536/23.74, 536/24.1, 536/24.2, 935/52, 935/55, 935/56, 935/57, 935/58, 935/69, 935/70, 935/71, 935/72, 935/22, 935/8
• US Class Current: 435/325
• 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 composition that effects recombination in mammalian cells comprising:

(i) an isolated and purified FLP recombinase, or an isolated and purified nucleotide sequence encoding same, and

(ii) a first DNA comprising a nucleotide sequence containing a first FLP recombination target site (FRT) therein,

wherein the genome of the mammalian cells contains a stably integrated second FRT site, and wherein said FLP recombinase catalyzes recombination between said first FRT and the second FRT, thereby precisely targeting integration of said first DNA into the genome.

2. A composition according to claim 1, wherein said first DNA comprises at least a first segment of a first gent of interest, said composition further comprising:

(iii) a second DNA comprising;

(a) at least a second segment of said first gene of interest, or

(b) at least a segment of a second gent of interest;

wherein said second DNA contains at least one FLP recombination target site; and

wherein said second DNA, when combined in reading frame with said first DNA, provides a functional gene.

3. A composition according to claim 2 wherein said second DNA comprises said second segment of said first gene of interest.

4. A composition according to claim 2 wherein said second DNA comprises said segment of said second gene of interest.

5. A composition according to claim 4 wherein said segment of said second gene of interest, when combined in reading frame with said first DNA, provides a hybrid, functional gene.

6. A composition according to claim 4 wherein said segment of said second gene of interest, when combined with said first DNA, disrupts the function of said first gene of interest.

7. A composition according to claim 1 wherein said first DNA further comprises a third FRT.

8. A composition according to claim 1 wherein said FLP recombinase is from a species of the genus Saccharomyces.

9. A composition according to claim 1 wherein said FLP recombinase is from a strain of Saccharomyces cerevisiae.

10. A composition according to claim 9 wherein said FLP recombinase is encoded by the sequence set forth as SEQ ID NO:

1.

11. A composition according to claim 1 wherein said first DNA provides a readily analyzable marker upon expression.

12. A composition according to claim 2 wherein said second DNA provides a readily analyzable marker upon expression.

13. A composition according to claim 1 wherein said first DNA is an autonomous fragment comprising:

(a) said first FRT,

(b) at least one restriction endonuclease recognition site,

(c) at least one marker gene,

(d) a bacterial origin of replication, and optionally

(e) a mammalian cellular or vital origin of DNA replication.

14. A DNA construct comprising, as an autonomous fragment, in the following order, reading from 5'"'"' to 3'"'"' along said fragment:

(a) a first FLP recombination target site,

(b) an insert segment comprising;

(1) at least one restriction endonuclease recognition site,

(2) at least one marker gene,

(3) a bacterial origin of replication, and optionally

(4) a mammalian cellular or viral origin of DNA replication, and

(c) a second FLP recombination target site in tandem with said first FLP recombination target site.

15. A method for the assembly of functional gene(s) for expression in mammalian cells, by recombination of individual inactive gene segments from one or more gene(s) of interest, wherein each of said segments contains at least one FRT, said method comprising:

contacting said individual inactive gene segments with a FLP recombinase, under conditions suitable for recombination to occur, thereby providing upon recombination a DNA which encodes a functional gene of interest, the expression product of which is biologically active.

16. A method according to claim 15 wherein said FLP recombinase is from a species of the genus Saccharomyces.

17. A method according to claim 15 wherein said FLP recombinase is from a strain of Saccharomyces cerevisiae.

18. A method according to claim 17 wherein said FLP recombinase is encoded by the sequence set forth as SEQ ID NO:

1.

19. An isolated mammalian cell, Wherein the genomic DNA of said cell contains an inserted heterologous first DNA comprising at least one FLP recombination target site therein.

20. An isolated mammalian cell according to claim 19 wherein said first DNA comprises at least a first segment of one or more first gene(s) of interest.

21. An isolated mammalian cell according to claim 20, further comprising DNA encoding, and capable of expressing, in said cell, a FLP recombinase.

22. An isolated mammalian cell according to claim 20 wherein said first gene(s) of interest provide a readily analyzable marker feature upon expression.

23. An isolated mammalian cell according to claim 19 wherein said FLP recombination target site has the sequence:

space="preserve" listing-type="equation">5'"'"'-GAAGTTCCTATTCTCTAGAAAGTATAGGAACTTC-3'"'"'.

24. An isolated mammalian cell according to claim 20 further comprising a second DNA, wherein said second DNA comprises:

(a) at least a second segment of said first gene of interest, or

(b) at least a segment of a second gene of interest;

wherein said second DNA contains at least one FLP recombination target site; and

wherein said second DNA, when combined in reading frame with said first DNA, provides a functional gene.