Gene targeting methods and vectors

• US 20040214222A1
• Filed: 05/19/2004
• Published: 10/28/2004
• Est. Priority Date: 06/26/2001
• Status: Abandoned Application

• Current Assignee: Genome Biosciences LLC
• Original Assignee: Genome Biosciences LLC
• Inventors: Burgess, Robert Marshall Jr., Ji, Henry Hongjun
• Application Number:
  US10/850,293
• Publication Number:
  US 20040214222A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2217/075 inducing loss of function, i.e. knock out
• CPC Class Codes: C12N 15/8213 Targeted insertion of genes into the plant genome by homologous recombination
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2830/42 being an intron or intervening sequence for splicing and/or stability of RNA

Abstract

Methods and vectors are provided for the specific alteration of particular genetic loci in eukaryotic cells. One method includes the utilization of positive-positive selection (PPS) DNA vectors for the purpose of creating and identifying cells which have vector sequences integrated into host cell genome via site-specific homologous recombination. The method also comprises the utilization of 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. The invention also includes vectors for creating modifications in eukaryotic cells.

Claims

1. A method for identifying a transformed cell which has undergone site-specific homologous recombination utilizing a PPS vector comprising:

a) transforming cells with a PPS vector designed to undergo site-specific homologous recombination wherein the vector comprises;

a first DNA sequence which is substantially homologous to an endogenous genomic sequence present within the host genome;

a second DNA sequence which encodes a positive selection characteristic in said cells and is non-homologous to cellular endogenous genomic sequences and therefore incapable of undergoing site-specific homologous recombination;

a third DNA sequence which is substantially homologous to an endogenous genomic sequence present within the host genome and is different from the first DNA sequence; and

a fourth DNA sequence which encodes a positive selection characteristic in said cells and is non-homologous to a cellular endogenous genomic sequence and therefore incapable of undergoing site-specific homologous recombination;

b) propagating cells to select for or enrich for those which have been transformed with said PPS vector by selecting for the presence of the positive selectable marker gene product of said second DNA sequence; and

c) separating cells which have said second DNA sequence encoding a positive selectable marker from cells which have said fourth DNA sequence encoding a positive selectable marker.

2. The method of claim 1, further comprising

d) characterizing the genomic DNA of said cells carrying the second DNA sequence encoding a positive selectable marker but not carrying the fourth DNA sequence encoding a positive selectable marker for the site-specific homologous recombination events which allow for modification of the cellular target DNA.

3. The method of claim 1 wherein said PPS vector includes positive selectable markers detectable by addition of antibiotics to cell cultures.

4. The method of claim 1 wherein said PPS vector includes positive selectable markers which may be detected by fluorescence light emission.

5. The method of claim 1 wherein said positive selectable markers allow for the separation of cells containing DNA encoding one marker from cells containing DNA encoding another or both markers.

6. The method of claim 1 wherein said cells are capable of homologous recombination.

7. The method of claim 1 wherein said cells are from a multicellular organism.

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 cells may be utilized to create a multicellular organism.

11. The method of claim 1 wherein said cells are embryonic stem cells.

25. An enriched population of cells generated through a method according to claim 1 wherein said cells have undergone site-specific homologous recombination.

26. A non-human transgenic animal generated by the method of claim 1 wherein said animal has been generated from cells which have undergone site-specific homologous recombination.

8. (Cancelled).

12. An isolated PPS vector for site-specific homologous recombination in cells capable of undergoing homologous recombination, the vector comprising:

a first DNA sequence which is substantially homologous to cellular endogenous genomic sequences and is capable of undergoing homologous recombination in said cells,

a second DNA sequence which is nonhomologous to cellular endogenous genomic sequences, is not capable of undergoing homologous recombination in said cells, and encodes a positive selectable marker capable of allowing for the identification of cells containing said positive selectable marker,

a third DNA sequence which is substantially homologous to cellular endogenous genomic sequences and is capable of undergoing homologous recombination in said cells,

a fourth DNA sequence which is nonhomologous to cellular endogenous genomic sequences, is not capable of undergoing homologous recombination in said cells, and encodes a positive selectable marker which allows for the separation of cells containing said positive selectable marker from cells not containing said positive selectable marker

wherein the organization of said PPS vector in 5′

to 3′

orientation comprises;

the first DNA sequence which is substantially homologous to cellular endogenous genomic DNA sequences, the second DNA sequence which encodes a positive selectable marker, the third DNA sequence which is substantially homologous to cellular endogenous genomic DNA sequences, and the fourth DNA sequence which encodes a positive selectable marker;

wherein the vector is capable of undergoing site-specific homologous recombination resulting in modification of cellular endogenous target genomic DNA sequences.

13. The PPS vector of claim 12 wherein said cellular endogenous genomic target DNA is comprised of exons and introns.

14. The PPS vector of claim 13 wherein said vector contains all or portions of exons and introns which are substantially homologous to cellular target genomic DNA sequences.

15. The PPS 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 PPS vector of claim 12 wherein said vector contains alterations in sequences which are substantially homologous to cellular target genomic DNA sequences including deletions, substitutions, additions or point mutations.

17. The PPS vector of claim 12 wherein said positive selection marker encoded by said fourth DNA sequence is selected from DNA sequences encoding fluorescent proteins including GFP, CFP, YFP, RFP, dsRED or HcRED.

18. The PPS vector of claim 12 wherein said positive selection marker encoded by said second DNA sequence is selected from a group of DNA sequences encoding resistance markers including neo, puro, blasticidin, bleomycin, zeocin or hygro.

19. The PPS vector of claim 12 wherein said positive selection marker encoded by said second DNA sequence is selected from a group of DNA sequences encoding fluorescent proteins including GFP, CFP, YFP, RFP, dsRED and HcRED.

20. The PPS vector of claim 12 wherein said vector may include an additional fifth DNA sequence which is nonhomologous to cellular endogenous genomic DNA sequences, is positioned external to said first and third DNA sequences on the opposite side containing said fourth DNA sequence which encodes a positive selectable marker and encodes a positive selectable marker.

21. The PPS vector of claim 12 wherein said fourth and fifth DNA sequences may encode positive selectable markers which allow for the separation of cells containing DNA encoding said selectable markers from cells which do not contain DNA encoding said selectable markers.

22. The PPS vector of claim 12 wherein said vector has lengths of homology for said first and third DNA sequences which are between about 50 bp and 50,000 base pairs.

23. The PPS vector of claim 12 wherein said vector results in the modification of cellular endogenous genomic target DNA sequences.

24. The PPS vector of claim 12 wherein said vector introduces exogenous regulatory elements into the cellular endogenous genomic target DNA sequences.

27-51. -51. (Cancelled).