Methods of performing homologous recombination based modification of nucleic acids in recombination deficient cells and use of the modified nucleic acid products thereof
AssigneeThe Rockfeller University
InventorsNathaniel Heintz, Peter Model, Xiangdong W. Yang
Life of this patent
Estimating the term…
Abstract
A simple method for modifying genes in a recombination deficient host cell is disclosed. Such modifications include generating insertion, deletions, substitutions, and/or point mutations at any chosen site in the independent origin based cloning vector. The modified gene can be contained in an independent origin based cloning vector that is used to introduce a modified heterologous gene into a cell. Such a modified vector may be used in the production of a germline transmitted transgenic animal, or in gene targeting protocols in eukaryotic cells.
Claims50
A method of selectively performing homologous recombination on a particular nucleotide sequence contained in a recombination deficient host cell comprising:
- (a) introducing a recombination cassette into the recombination deficient host cell, wherein the recombination cassette contains a nucleic acid that selectively integrates into the particular nucleotide sequence when the host cell is induced to support homologous recombination, and wherein the host cell cannot independently support homologous recombination;
- and
- (b) inducing the host cell to transiently support homologous recombination wherein the nucleic acid integrates into the particular nucleotide sequence;
- and wherein unselected nucleotide sequence rearrangements and deletions which are indicative of host cells that support homologous recombination, are not evident with a restriction endonuclease digestion map analysis with HindIII, EcoRI, XhoI, or AvrII.
A conditional replication shuttle vector that encodes a RecA-like protein.
An independent origin based cloning vector that contains a nucleic acid that has been directly modified with specificity by having undergone homologous recombination with a conditional replication shuttle vector in a RecA.sup.- host cell, wherein the conditional replication shuttle vector encodes a RecA-like protein.
A kit for performing homologous recombination in a BBPAC comprising:
- (a) a conditional replication shuttle vector;
- (b) a building vector;
- (c) a restriction map for the shuttle vector;
- and
- (d) a restriction map for the building vector.
A kit for performing homologous recombination in a BBPAC comprising:
- (a) the pSV1.RecA temperature-sensitive shuttle vector;
- and
- (b) a building vector selected from the group consisting of pBV.IRES.LacZ.PA;
- pBV.EGFP1;
- pBV.IRES.EGFP1;
- and pBV.pGK.Neo.PA.
An independent origin based cloning vector that contains a nucleic acid that has been directly modified with specificity by having undergone homologous recombination with a conditional replication shuttle vector in a RecA.sup.- host bacterial cell, wherein the conditonal replication shuttle vector encodes a protein that can support homologous recombination in the RecA.sup.- host bacterial cell.
Dependencies are inferred from the claim text.