Enhanced homologous recombination mediated by lambda recombination proteins

Abstract

Disclosed herein are methods for generating recombinant DNA molecules in cells using homologous recombination mediated by recombinases and similar proteins. The methods promote high efficiency homologous recombination in bacterial cells, and in eukaryotic cells such as mammalian cells. The methods are useful for cloning, the generation of transgenic and knockout animals, and gene replacement. The methods are also useful for subcloning large DNA fragments without the need for restriction enzymes. The methods are also useful for repairing single or multiple base mutations to wild type or creating specific mutations in the genome. Also disclosed are bacterial strains and vectors which are useful for high-efficiency homologous recombination.

1 . A method of altering a eukaryotic nucleic acid sequence, comprising:
 introducing a nucleic acid of interest into a host cell, wherein the host cell comprises an extrachromosomal element comprising at least one intron, at least one exon, or both an intron and an exon of the eukaryotic nucleic acid sequence, wherein the nucleic acid of interest is a single stranded nucleic acid in length or wherein the nucleic acid of interest is a double stranded DNA comprising a single stranded overhang, and wherein the nucleic acid of interest comprises a sufficient number of nucleotides homologous to the eukaryotic nucleic acid sequence to mediate homologous recombination, and wherein the nucleic acid of interest is at least 30 nucleotides in length, and wherein the host cell comprises a pL promoter operably linked to a nucleic acid sequence encoding a recombinase; and   activating expression of the recombinase from the de-repressible promoter; wherein expression of the recombinase results in the nucleic acid of interest undergoing homologous recombination with the eukaryotic nucleic acid sequence,   thereby altering the eukaryotic nucleic acid sequence in the extrachromosmal element.   
     
     
         2 . The method of  claim 1 , wherein the recombinase is a single-stranded DNA binding protein. 
     
     
         3 . The method of  claim 1 , wherein the recombinase is a double-strand break repair recombinase. 
     
     
         4 . The method of  claim 1 , wherein the DNA encoding the recombinase encodes lambda Beta. 
     
     
         5 . The method of  claim 4 , wherein the DNA encoding the nucleic acid sequence further encodes lambda Exo. 
     
     
         6 . The method of  claim 4 , wherein the DNA encoding the recombinase further encodes lambda Gam. 
     
     
         7 . The method of  claim 1 , wherein the extachromosomal element is a bacterial artificial chromosome, a yeast artificial chromosome, a P1 artificial chromosome, a plasmid, or a cosmid. 
     
     
         8 . The method of claim  61 , wherein the eukaryotic nucleic acid sequence is a mammalian nucleic acid sequence. 
     
     
         9 . The method of  claim 1 , wherein the recombinase is lambda Beta, and wherein the pL promoter operably linked to the nucleic acid sequence encoding the recombinase is a mini-lambda. 
     
     
         10 . The method of  claim 1 , wherein the host cell is a bacterial cell. 
     
     
         11 . The method of  claim 10 , and wherein the bacterial cell comprises a lambda prophage, and wherein the lambda prophage does not comprise cro through attR, and does not comprise bioA, and wherein the prophage comprises a selectable marker substituted for a cro-bioA segment. 
     
     
         12 . The method of  claim 10 , wherein the bacterial cell is RecA-. 
     
     
         13 . The method of  claim 1 , wherein the selectable marker confers tetracycline resistance to the bacterial cell. 
     
     
         14 . The method of  claim 1 , wherein the nucleic acid of interest encodes an epitope tag. 
     
     
         15 . A method of subcloning a DNA sequence, comprising:
 providing a bacterial cell comprising a nucleic acid sequence encoding lambda Exo, Beta and Gam operably linked to pL, and wherein the bacterial cell further comprises a DNA molecule with a target nucleic acid sequence, wherein the DNA molecule is located extrachromosomally;   introducing into the bacterial cell a linear plasmid vector with a 5′ and a 3′ end, wherein the linear plasmid vector comprises an origin of replication, wherein the linear plasmid vector comprises a nucleotide sequence homologous to the target nucleic acid sequence on the 5′ end and a nucleic acid sequence homologous to the target nucleic acid sequence on the 3′ end;   inducing the expression of the nucleic acid encoding Exo, Beta and Gam, thereby inducing homologous recombination between the linear plasmid vector and the target nucleic acid sequence; thereby inserting the target nucleic acid into the linear plasmid vector, thereby forming a circularized plasmid vector; and   isolating the circularized plasmid vector, wherein the circularized plasmid vector comprises a selectable marker,   thereby subcloning the DNA sequence.   
     
     
         16 . The method of  claim 15 , wherein the DNA molecule comprises at least about 20 kb of DNA. 
     
     
         17 . The method of  claim 15 , wherein the DNA molecule comprises at least about 40 kb of DNA. 
     
     
         18 . The method of  claim 15 , wherein the DNA comprises at least about 80 kb of DNA. 
     
     
         19 . The method of  claim 15 , wherein the extrachromosomal DNA is a bacterial artificial chromosome. 
     
     
         20 . The method of  claim 15 , wherein the bacterial cell is an  E. coli  cell. 
     
     
         21 . The method of  claim 15 , wherein the  E. coli  cell is a recBC sbcC strain. 
     
     
         22 . The method of  claim 15 , wherein the nucleotide sequence homologous to the target nucleic acid sequence on the 5′ end and the nucleotide sequence homologous to the target nucleic acid sequence on the 3′ end are single stranded. 
     
     
         23 . The method of  claim 20 , wherein the  E. coli  cell comprises a lambda prophage, and wherein the lambda prophage does not comprise cro through attR, and does not comprise bioA, and wherein the prophage comprises a selectable marker substituted for a cro-bioA segment. 
     
     
         24 . The method of  claim 15 , wherein the DNA sequence encodes a therapeutic protein. 
     
     
         25 . The method of  claim 15 , wherein a human chromosomal nucleic acid sequence comprises the target nucleic acid sequence. 
     
     
         26 . The method of  claim 1 , wherein the eukaryotic nucleic acid sequence encodes a therapeutic protein. 
     
     
         27 . The method of  claim 1 , wherein the eukaryotic nucleic acid sequence is a human nucleic acid sequence.