Gene targeting methods and vectors

• US 20030135872A1
• Filed: 12/03/2002
• Published: 07/17/2003
• Est. Priority Date: 12/04/2001
• Status: Abandoned Application

• Current Assignee: Genome Biosciences LLC
• Original Assignee: Genome Biosciences LLC
• Inventors: Burgess, Robert M. Jr., Ji, Henry H.
• Application Number:
  US10/309,757
• Publication Number:
  US 20030135872A1
• Time in Patent Office: Days
• US Class Current: 800/8
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/907 in mammalian cells

Abstract

Procedures and vectors are provided for the specific alteration of particular genetic loci in eukaryotic cells. One procedure utilizes fluorescence-probe-in-cell (FPIC) gene targeting DNA vectors for the purpose of creating and identifying cells which have vector sequences integrated into the host cell genome via site-specific homologous recombination. The procedure also utilizes sequences encoding in vivo detectable markers for the identification of cells which have exogenous vector sequences integrated into the genome of the host cell, either via site-specific homologous recombination or nonhomologous recombination or insertion. In addition, cells modified using a FPIC vector, and organisms generated from such cells, are provided.

Claims

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.

35-1. A non-human transgenic animal generated from a cell of claim 35.