Claims
- 1. A method for obtaining a transformed cell that has undergone site-specific homologous recombination utilizing a FPIC gene targeting vector, comprising:
a) contacting cells with an FPIC gene targeting vector designed to undergo site-specific homologous recombination, under conditions suitable for transformation of the cells by the vector, wherein the vector comprises:
a first DNA sequence that is substantially homologous to cellular endogenous genomic sequences and is capable of undergoing homologous recombination in said cells, a second DNA sequence that is not homologous to cellular endogenous genomic sequences, and is not capable of undergoing homologous recombination in said cells, said second DNA sequence further comprising a nucleotide sequence that allows identification of cells containing said nucleotide sequence, a third DNA sequence that is substantially homologous to cellular endogenous genomic sequences and is capable of undergoing homologous recombination in said cells, a fourth DNA sequence that is not homologous to cellular endogenous genomic sequences, and is not capable of undergoing homologous recombination in said cells, said fourth DNA sequence further comprising a nucleotide sequence capable that allow identification and isolation of cells containing said nucleotide sequences from cells not containing said nucleotide sequence; b) propagating the cells under conditions selective for the presence of transcribed sequences representing said second DNA sequence and for the absence of transcribed sequences representing said fourth DNA sequence, thereby selecting or enriching for cells transformed with said FPIC gene targeting vector; and c) separating cells having said second DNA sequence from cells having said fourth DNA, thereby obtaining transformed cells that have undergone site-specific homologous recombination utilizing a FPIC gene targeting vector.
- 2. The method of claim 1, further comprising characterizing the genomic DNA of said transformed cells carrying the second DNA sequence encoding a positive selectable marker but not carrying the fourth DNA sequence encoding a transcribed selection characteristic for the site-specific homologous recombination events which allow for modification of the cellular target DNA
- 3. The method of claim 1 wherein said FPIC gene targeting vector comprises a positive selectable marker, which is detectable by incubating said cells in the presence of an antibiotic, or by fluorescence light emission.
- 4. The method of claim 1 wherein said FPIC gene targeting vector comprises a negative selectable marker, which is detectable by incubating said cells in the presence of an antibiotic.
- 5. The method of claim 1 wherein said FPIC gene targeting vector comprises a negative selectable marker, which is detectable without using an antibiotic or drug.
- 6. The method of claim 1, wherein said cells are capable of homologous recombination.
- 7. The method of claim 1, wherein said cells are cells of a multicellular organism.
- 8. The method of claim 1, wherein said cells are plant cells.
- 9. The method of claim 1, wherein said cells have undergone multiple rounds of site-specific homologous recombination for the purposes of multiple modifications of the endogenous cellular genome.
- 10. The method of claim 1, wherein said transformed cells are utilized to generate a multicellular organism.
- 11. The method of claim 1, wherein said cells are embryonic stem cells.
- 12. An isolated fluorescence-probe-in-cell (FPIC) gene targeting vector for site-specific homologous recombination in cells capable of undergoing homologous recombination, the vector comprising:
a first DNA sequence that is substantially homologous to cellular endogenous genomic sequences and is capable of undergoing homologous recombination in said cells, a second DNA sequence that is not homologous to cellular endogenous genomic sequences, and is not capable of undergoing homologous recombination in said cells, said second DNA sequence further comprising a nucleotide sequence capable of allowing identification of cells containing said nucleotide sequence, a third DNA sequence that is substantially homologous to cellular endogenous genomic sequences and is capable of undergoing homologous recombination in said cells, a fourth DNA sequence that is not homologous to cellular endogenous genomic sequences, and is not capable of undergoing homologous recombination in said cells, said fourth DNA sequence further comprising a nucleotide sequence capable of allowing identification and separation of cells containing said DNA sequences from cells not containing said nucleotide sequence. said FPIC gene targeting vector comprising, in 5′ to 3′ orientation, the first DNA sequence that is substantially homologous to cellular endogenous genomic DNA sequences, the second DNA sequence, the third DNA sequence that is substantially homologous to cellular endogenous genomic DNA sequences, and the fourth DNA sequence; and wherein said vector is capable of undergoing site-specific homologous recombination resulting in modification of cellular endogenous target genomic DNA sequences.
- 13. The FPIC gene targeting vector of claim 12, wherein said cellular endogenous genomic target DNA comprises exons and introns.
- 14. The FPIC gene targeting vector of claim 13, wherein said vector contains all or portions of said exons and introns, which are substantially homologous to cellular target genomic DNA sequences.
- 15. The FPIC gene targeting vector of claim 12, wherein said vector contains all or portions of regulatory elements, which are substantially homologous to cellular target genomic DNA sequences.
- 16. The FPIC gene targeting vector of claim 12, wherein said vector contains alterations in the sequences that are substantially homologous to cellular target genomic DNA sequences, said alterations comprising deletions, substitutions, additions, point mutations, or a combination thereof.
- 17. The FPIC gene targeting vector of claim 12, wherein said second DNA sequence encodes a transcribed nonfunctional selection characteristics in said cells and is non-homologous to a cellular endogenous genomic sequence and therefore incapable of undergoing site-specific homologous recombination.
- 18. The FPIC gene targeting vector of claim 12, wherein said fourth DNA sequence encodes a transcribed nonfunctional selection characteristics in said cells and is non-homologous to a cellular endogenous genomic sequence and therefore incapable of undergoing site-specific homologous recombination.
- 19. The FPIC gene targeting vector of claim 12, wherein said fourth DNA sequence comprises a nucleotide sequence encoding a fluorescent protein.
- 20. The FPIC gene targeting vector of claim 19, wherein the fluorescent protein is GFP, CFP, YFP, RFP, dsRED, or HcRED.
- 21. The FPIC gene targeting vector of claim 12, wherein said second DNA sequence comprises a nucleotide sequence encoding an antibiotic resistance marker.
- 22. The FPIC gene targeting vector of claim 21, wherein said antibiotic resistance marker is neomycin, puromycin, blasticidin, bleomycin, zeocin, or hygromycin.
- 23. The FPIC gene targeting vector of claim 12, wherein said second DNA sequence comprises a nucleotide sequence encoding a fluorescent protein.
- 24. The FPIC gene targeting vector of claim 23, wherein the fluorescent protein is GFP, CFP, YFP, RFP, dsRED, or HcRED.
- 25. The FPIC gene targeting vector of claim 12, wherein said fourth DNA sequence comprises a nucleotide sequence encoding a negative selection agent.
- 26. The FPIC gene targeting vector of claim 25, wherein the negative selection agent is thymidine kinase or diphtheria toxin A chain.
- 27. The FPIC gene targeting vector of claim 12, wherein said vector further comprises a fifth DNA sequence that is not homologous to cellular endogenous genomic DNA sequences, and is positioned external and 5′ to said first and third DNA sequences.
- 28. The FPIC gene targeting vector of claim 12, wherein each of said fourth and fifth DNA sequences encode a selectable transcribed sequence that allows for separation of cells containing DNA encoding said selectable transcribed sequences from cells that do not contain DNA encoding said selectable transcribed sequences.
- 29. The FPIC gene targeting vector of claim 12, wherein the substantially homologous sequence of said first DNA sequence and said third DNA sequence each is about 50 base pairs to 50,000 base pairs.
- 30. A cell transformed by the FPIC gene targeting vector of claim 12.
- 31. The cell of claim 30, wherein said vector results in the modification of at least one cellular endogenous genomic target DNA sequence.
- 32. The cell of claim 31, wherein said vector introduces at least one exogenous regulatory element into the cellular endogenous genomic target DNA sequence.
- 33. The cell of claim 30, which is an embryonic stem cell.
- 34. A transgenic non-human animal generated from the cell of claim 31.
- 35. An enriched population of cells generated by the method of claim 1, wherein said cells have undergone site-specific homologous recombination.
- 35. A non-human transgenic animal generated from a cell of claim 35.
- 36. A transgenic plant generated from a cell of claim 35.
Parent Case Info
[0001] The present application claims the benefit priority under 35 U.S.C. §119(e) to U.S. Serial No. 60/338,768, filed Dec. 4, 2001, the entire contents of which is incorporated herein by reference.
Provisional Applications (1)
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Number |
Date |
Country |
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60338768 |
Dec 2001 |
US |