Method of constructing vectors for homologous recombination directed mutagenesis

Abstract

The present invention provides a novel vector system and thereby a novel method for the simplified construction of recombinant vectors for directed mutagenesis. Said vector system is used to modify the eukaryotic genome, particularly of embryonic stem cells, at precise and predefined loci by the means of homologous recombination. Further more, said system finds its usage in the generation of new strategies for gene therapy and in the generation of genetically modified higher eukaryotic organisms.

What is claimed is:  
     
         1 . An insertional mutagenesis system useful in constructing vectors for targeted mutagenesis of mammalian cells by homologous recombination, said insertional mutagenesis system comprising: 
 (a) a linear lambda vector for cloning of genomic DNA flanked by negative selection markers; and    (b) a vector for insertion of a positive selection cassette into cloned genomic DNA.    
     
     
         2 . The insertional mutagenesis system of  claim 1 , said linear lambda vector comprising: 
 (a) a stuffer fragment of DNA flanked by restriction endonuclease sequences to facilitate the cloning of genomic DNA;    (b) an  E.coli  origin of replication;    (c) an antibiotic resistance gene;    (d) a yeast origin of replication;    (e) a selectable marker suitable for use in yeast;    (f) a negative selectable marker(s) including a promoter element operatively positioned 5′ to said negative selectable marker and a polyadenylation site operatively positioned 3′ to said negative selectable marker.    (g) a direct repeat of recombinase sequences for recombinase directed conversion of the linear lambda phage vector into an  E. coli /yeast shuttle plasmid.    
     
     
         3 . The insertional mutagenesis system of  claim 2 , wherein said recombinase sequence and corresponding recombinase are selected from the group consisting of LoxP sequences-Cre recombinase and Frt sequences-Flp recombinase.  
     
     
         4 . The insertional mutagenesis system of  claim 1 , said vector for the insertion of a positive selection cassette comprises: 
 (a) an  E.coli  origin of replication;    (b) an antibiotic resistance gene;    (c) a selectable marker suitable for use in yeast;    (d) a positive selectable marker, wherein a promoter element is operatively positioned 5′ to said positive selectable marker and a polyadenylation site operatively positioned 3′ to said positive selectable marker;    (e) unique restriction endonuclease sequences flanking the positive selectable marker so that said marker can be exchanged for another positive selection marker;    (f) unique restriction endonuclease sequences for the excision of the positive selective cassette prior to ligation of synthetic deoxynucleotides to the cassette ends;    (g) restriction endonuclease sequences flanking the bacterial and yeast sequences to facilitate the removal of these sequences from the vector.    
     
     
         5 . A method of generating mutations at specific sites in cloned genomic DNA, comprising the steps of: 
 (a) cloning genomic DNA into the linear lambda vector of  claim 1;     (b) isolating a clone of interest;    (c) converting the linear lambda vector containing the genomic DNA of interest into a circular  E. coli /yeast shuttle vector by infecting a Cre recombinase expressing bacterial strain;    (d) identifying genomic DNA sequences intended for targeting of the positive selection cassette;    (e) synthesizing deoxyoligonucleotides complementary to sequences flanking the site intended for targeting of the positive selection cassette to said circular  E. coli /yeast shuttle vector;    (f) attaching said synthetic deoxyoligonucleotides to the positive selection cassette by ligation or PCR;    (g) co-transforming said  E. coli /yeast shuttle vector containing the genomic DNA of interest and the modified positive selection cassette into a yeast host cell, wherein an intact recombinant plasmid is selected for by means of the gene products provided for by yeast selectable markers and is obtained by performing homologous DNA recombination between homologous regions of the vector containing genomic DNA and the synthetically derived sequences which had been ligated onto the ends of the positive selection cassette thereby generating a new recombinant vector with the positive selection cassette inserted into the genomic DNA sequences.    
     
     
         6 . The method of generating mutations at specific sites in cloned genomic DNA of  claim 5 , wherein the  E. coli /yeast sequences of the positive selection cassette vector are removed.  
     
     
         7 . The method of  claim 6 , wherein said removal comprises: 
 (a) digesting said recombinant vector with the positive selection cassette inserted into the genomic DNA sequences with restriction endonuclease specific for the unique restriction endonuclease sequences incorporated into the positive selection cassette vector which flank the  E. coli /yeast sequences in said vector;    (b) ligating said digested vector; and    (c) identifying ligation products lacking the  E.coli /yeast sequences desired to be removed.    
     
     
         8 . A method of producing mutated animal cells, comprising the steps of: 
 (a) linearizing said recombinant vector having a positive selection cassette inserted into the genomic DNA sequences at a unique restriction endonuclease sequence outside the sequences of the cloned genomic DNA;    (b) introducing said linearized DNA into an animal cell;    (c) selecting transduced cells that express said positive selectable marker; and    (d) selecting transduced cells that express said positive selectable marker and do not express said negative marker.    
     
     
         9 . The method of  claim 8 , wherein said animal cell is an embryonic stem cell or any other cell type with the potential to generate an animal.  
     
     
         10 . A method for the production of a non-human transgenic animal consisting of: 
 (a) introduction of said mutated animal cells in  claim 8  into animal embryos;    (b) placement of the embryos containing said mutated cells into the uterus of a female animal; and    (c) replacement of the nucleus of a fertilized egg with the nucleus containing the modified genomic DNA.