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

• US 6,956,146 B2
• Filed: 02/28/2002
• Issued: 10/18/2005
• Est. Priority Date: 03/08/1991
• Status: Expired due to Fees

• Current Assignee: Corn Products International Incorporated (Ingredion Incorporated)
• Original Assignee: Salk Institute for Biological Studies
• Inventors: Wahl, Geoffrey M., O'Gorman, Stephen V.
• Primary Examiner(s): Woitach, Joseph
• Assistant Examiner(s): BERTOGLIO, VALARIE E
• Application Number:
  US10/086,542
• Publication Number:
  US 20020166138A1
• Time in Patent Office: 1,328 Days
• Field of Search: 800/14, 800/18, 800/13, 800/15, 435/455, 435/462
• US Class Current: 800/14
• 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 transgenic, non-human mammal, wherein said mammal contains at least one FLP recombination target site in the genomic DNA thereof, wherein said FLP recombination target site comprises the 13 base-pair repeats of SEQ ID NO:

3, separated by an 8 base-pair spacer.

2. A transgenic, non-human mammal according to claim 1, wherein said FLP recombination target site is positioned within at least a portion of a first gene of interest.

3. A transgenic, non-human mammal according to claim 1, further comprising a nucleotide sequence encoding, and capable of being expressed, in transgenic, non-human mammals, an FLP recombinase.

4. A transgenic, non-human mammal according to claim 1, further comprising FLP recombinase.

5. A transgenic, non-human mammal according to claim 1, wherein said mammal is a mouse.

6. A transgenic, non-human mammal according to claim 1, wherein said mammal is a rat.

7. A transgenic, non-human mammal according to claim 1, wherein said mammal is a monkey.

8. A transgenic, non-human mammal according to claim 1, wherein said mammal is a hamster.

9. A transgenic, non-human mammal according to claim 2, wherein said first gene of interest provides a readily analyzable marker feature to the host system.

10. A transgenic, non-human mammal according to claim 9, wherein said marker is selected from the group consisting of β

-galactosidase, thymidine kinase;

tyrosinase, and antibiotic resistance.

11. A transgenic, non-human mammal according to claim 2, further comprising an additional DNA fragment, wherein said additional DNA fragment is selected from:

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

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

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

wherein said additional DNA fragment, when combined in reading frame with said at least a portion of said first gene of interest, provides a functional gene.

12. A transgenic, non-human mammal according to claim 2, further comprising an additional DNA fragment, wherein said additional DNA fragment is selected from:

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

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

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

wherein site-specific recombination disrupts the function of said first gene of interest.

13. A transgenic, non-human mammal according to claim 12, wherein said function is colorimetrically detectable, immunologically detectable or genetically detectable.

14. A transgenic, non-human mammal according to claim 3, wherein said mammal is selected from the group consisting of a mouse, a rat, a monkey and a hamster.

15. A transgenic, non-human mammal according to claim 4, wherein said mammal is selected from the group consisting of a mouse, a rat, a monkey and a hamster.

16. A transgenic, non-human mammal according to claim 9, wherein said mammal is selected from the group consisting of a mouse, a rat, a monkey and a hamster.

17. A transgenic, non-human mammal according to claim 11, wherein said mammal is selected from the group consisting of a mouse, a rat, a monkey and a hamster.

18. A transgenic, non-human mammal according to claim 12, wherein said mammal is selected from the group consisting of a mouse, a rat, a monkey and a hamster.