Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-04-17
2002-10-22
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S320100, C435S455000, C435S465000
Reexamination Certificate
active
06468754
ABSTRACT:
FIELD OF THE INVENTION
This invention is related generally to the fields of recombinant DNA technology and genomics. More specifically, it is related to gene targeting vectors and methods for rapidly removing or altering a DNA sequence integrated into mammalian cells or host mammalian organisms, and methods for selecting cells using the methods of the invention.
BACKGROUND OF THE INVENTION
It is possible to modify a mammalian genome by adding. genetic material, but to further modify the introduced genetic material without causing additional alterations in the remaining genome has been a laborious and time consuming process. A system that would allow the simultaneous deletion of the introduced DNA and/or replacement of the introduced DNA would allow the researcher to monitor both the baseline conditions (deletion state) and any altered states of the inserted DNA in the same genetic background.
The ability to make such site specific alterations, deletions and insertions to transgenic cell lines has been described using various site specific recombinases paired with their DNA recognition sequences, such as Cre-lox or Flp-Frt (S. Fukushige et al.,
Proc Natl Acad Sci USA
(1992)89(17):7905-09; S. O'Gorman, et al.,
Science
(1991) 251:1351-35; B. Sauer et al.,
Proc Natl Acad Sci USA
(1988) 85(14):5166-70; B. Sauer et al.,
Nuc Acids Res
(1989) 17(1):147-61; B. Sauer et al.,
New Biol
(1990) 2(5):441-49. However the methods employed by these workers only allowed one type of change to be made in the introduced DNA, either an insertion or a deletion. Furthermore, the methods employed by these authors to detect and characterize the recombinase formed products are very time consuming and laborious.
SUMMARY OF THE INVENTION
We have now invented a vector system and method that facilitates insertion of a query gene into a eukaryotic host cell, and the subsequent removal, insertion, and/or substitution of a different query gene and/or marker gene at the same site within the host cell. One aspect of the invention is a polynucleotide vector, comprising in order of transcription: a regulatable promoter; a first recombinase target site; a second recombinase target site different from said first recombinase target site; a cloning site suitable for insertion of a test gene; an internal ribosome binding site (IRES); an optically-active marker-encoding sequence; a third recombinase target site homologous to either said first recombinase target site or said second recombinase target site. A presently-preferred subgenus is the vector further comprising a second promoter, and a selectable marker operatively associated with said second promoter.
Another aspect of the invention is a method of selecting a host cell having a functioning test gene, comprising: providing a host cell lacking a functioning test gene; inserting into said host cell a vector, said vector comprising a regulatable promoter; a first recombinase target site; a second recombinase target site different from said first recombinase target site; a test gene; an internal ribosome binding site (IRES); a label sequence encoding a detectable marker; and a third recombinase target site homologous to either said first recombinase target site or said second recombinase target site; selecting against cells that failed to incorporate said vector; inducing said regulatable promoter; and selecting for cells that express said detectable marker. A presently preferred sub-genus is the method further comprising: contacting said host cell with a recombinase capable of catalyzing excision of said label sequence.
Another aspect of the invention is the method of altering a host cell comprising a vector of the invention, said method comprising providing a host cell comprising a vector of the invention, and contacting said cell with an effective amount of a recombinase that recognizes said first recombinase target site or said second recombinase target site, such that the portion of the vector between the recombinase target sites is deleted. A presently preferred class of the invention is the method wherein said host cell is contacted with recombinase by intracellular expression of said recombinase. Another aspect of the invention is the method of substituting a query gene and/or marker gene in a host cell, comprising providing a host cell comprising a vector of the invention, and inserting into said cell a polynucleotide comprising a recombinase target site complementary to either said first or second recombinase target site along with an effective amount of a recombinase that recognizes said recombinase target, such that the portion of the vector between the recombinase target sites is replaced with the polynucleotide portion between the two recombinase target sites.
REFERENCES:
patent: 6025192 (2000-02-01), Beach et al.
Masuda et al., Effects of procollagen C-proteinase enhancer protein on the growth of cultured rat fibroblasts revealed by an excisable retroviral vector, 1998, Cell Growth & Differentiation, vol. 9, pp. 381-391.*
Fukushige et al., “Genomic Targeting With a Positive-Selection lox Integration Vector Allows Highly Reproduciable Gene Expression in Mammalian Cell,”Proc. Natl. Acad. Sci. U.S.A. 89(17):7905-7909 (1992).
O'Gorman et al., “Recombinase-Mediated Gene Activation and Site-Specific Integration in Mammalian Cells,”Science251:1351-1335 (1991).
Sauer et al., “Cre-Stimulated Recombination at loxP-Containing DNA Sequences Placed into the Mammalian Genome,”Nucleic Acids Research. 17(1):147-161 (1989).
Sauer et al., “Site-Specific DNA Recombination in Mammalian Cells by the Cre Recombinase of Bacteriophage P1,”Proc. Natl. Acad. Sci. U.S.A. 85(14):5166-5170.
Sauer et al., “Targeted Insertion of Exogenous DNA into the Eukaryotic Genome by the Cre Recombinase,”The New Biologist2(5):441-449 (1990).
Bouhassira et al., “Transcriptional Behavior of LCR Enhancer Elements Integrated at the Same Chromosomal Locus by Recombinase-Mediated Cassette Exchange,”Blood90(9):3332-3344 (1997).
Feng et al., “Site-Specific Chromosomal Integration in Mammalian Cells: Highly Efficient CRE Recombinase-Mediated Cassette Exchange,”Journal of Molecular Biology292(4):779-785 (1999).
Kirchoff et al., “Identification of Mammalian Cell Clones Exhibiting Highly Regulated Expression from Inducible Promoters,”Trends in Genetics11(6):219-220 (1995).
Metzger et al., “Conditional Site-Specific Recombination in Mammalian Cells Using a Ligand-Dependent Chimeric CRE Recombinase,”Proc. Natl. Acad. Sci. U.S.A. 92(15):6991-6995 (1995).
Seibler et al., “Double-Reciprocal Crossover Mediated by FLP-Recombinase: A Concept and an Assay,”Biochemistry, American Chemical Society36(7):1740-1747 (1997).
Seibler et al., “DNA Cassette Exchange in ES Cells Mediated by FLP Recominase: An Efficient Strategy for Repeated Modification of Tagged LOCI by Marker-Free Constructs,”Biochemistry37:6229-6234 (1998).
Snaith et al., “Multiple Cloning Sites Carrying LOXP and FRT Recognition Sites for the CRE and FIP Site-Specific Recominases,”Gene166(1):173-174 (1995).
Zhang et al., “Inducible Site-Directed Recombination in Mouse Embryonic Stem Cells,”Nucleic Acids Research24(4):543-548 (1996).
Greene Amy L.
Jarnigan Kurt
Thode Silke
Zhou Hua
Iconix Pharmaceuticals Inc.
Ketter James
Robins & Pasternak LLP
Sullivan Daniel
LandOfFree
Vector and method for targeted replacement and disruption of... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Vector and method for targeted replacement and disruption of..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Vector and method for targeted replacement and disruption of... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2987587