Methods of preforming homologous recombination based modification of nucleic acids in recombination deficient cells and use of the modified nucleic acid products thereof

Abstract

A simple method for modifying genes in a recombination deficient host cell is disclosed. Such modifications include generating insertions, deletions, substitutions, and/or point mutations at any chosen site in the independent origin based cloning vector. The modified gene is 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. In particular, high throughput methodology is provided for generating the modified the independent origin based cloning vectors of the present invention.

1 . A conditional replication shuttle vector comprising: 
 (a) an R6K origin of replication; and    (b) a nucleic acid encoding a recombination protein.    
     
     
         2 . The conditional replication shuttle vector of  claim 1  wherein the recombination protein is recA  
     
     
         3 . The conditional replication shuttle vector of  claim 1  further comprising a nucleic acid encoding a marker protein.  
     
     
         4 . The conditional replication shuttle vector of  claim 3  wherein the nucleic acid encoding the marker protein is IRES-EGFP.  
     
     
         5 . The conditional replication shuttle vector of  claim 3  further comprising a second marker protein.  
     
     
         6 . The conditional replication shuttle vector of  claim 5  wherein the nucleic acid encoding the second marker protein is taulacZ.  
     
     
         7 . The conditional replication shuttle vector of  claim 1  further comprising a gene that can be counter-selected against.  
     
     
         8 . The conditional replication shuttle vector of  claim 7  wherein the gene that can be counter-selected against is SacB.  
     
     
         9 . The conditional replication shuttle vector of  claim 7  wherein the gene that can be counter-selected against confers tetracycline resistance.  
     
     
         10 . The conditional replication shuttle vector of  claim 1  further comprising an A box region bracketed by two restriction enzyme sites; wherein said A box region and said restriction enzyme sites can be used to insert a selected nucleic acid into said conditional replication shuttle vector.  
     
     
         11 . The conditional replication shuttle vector of  claim 10  wherein the two restriction enzyme sites are Asc1 and Sma1.  
     
     
         12 . The conditional replication shuttle vector of  claim 1  further comprising two FRT sites; wherein the two FRT sites are on opposite sides of the A box.  
     
     
         13 . The conditional replication shuttle vector of  claim 1  further comprising two homologous nucleotide sequences of 500 basepairs or more; wherein the two homologous nucleotide sequences are on opposite sides of the A box.  
     
     
         14 . The conditional replication shuttle vector of  claim 13  wherein the two homologous nucleotide sequences encode the enhanced green fluorescent protein (EGFP).  
     
     
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         33 . An IOBCV that comprises a particular nucleotide sequence that has been modified by a method comprising the steps of: 
 (a) introducing a conditional replication shuttle vector into a recombination deficient host cell; wherein the host cell comprises an IOBCV that comprises a gene of interest which contains the particular nucleotide sequence; wherein the conditional replication shuttle vector encodes a recombination protein that is expressed by the host cell and permits homologous recombination to occur in the host cell; wherein the conditional replication shuttle vector contains a nucleic acid that selectively integrates into the particular nucleotide sequence when the recombination protein is expressed forming a co-integrate; wherein the nucleic acid that selectively integrates into the particular nucleotide sequence and the nucleic acid encoding the recombination protein are positioned on the conditional replication shuttle vector such that upon resolution of the co-integrate, the nucleic acid encoding the recombination protein remains with the conditional replication shuttle vector; and wherein neither the IOBCV alone, nor the IOBCV in combination with the host cell can independently support homologous recombination; and    (b) growing the host cell under conditions in which the conditional replication shuttle vector cannot replicate, therein diluting out the conditional replication shuttle vector encoding the recombination protein, and thereby preventing further recombination events in the recombination deficient cells.    
     
     
         34 . A BAC that comprises a particular nucleotide sequence that has been modified by a method comprising the steps of: 
 (a) introducing a conditional replication shuttle vector into a recombination deficient host cell; wherein the host cell comprises an IOBCV that comprises a gene of interest which contains the particular nucleotide sequence; wherein the conditional replication shuttle vector encodes a recombination protein that is expressed by the host cell and permits homologous recombination to occur in the host cell; wherein the conditional replication shuttle vector contains a nucleic acid that selectively integrates into the particular nucleotide sequence when the recombination protein is expressed forming a co-integrate; wherein the nucleic acid that selectively integrates into the particular nucleotide sequence and the nucleic acid encoding the recombination protein are positioned on the conditional replication shuttle vector such that upon resolution of the co-integrate, the nucleic acid encoding the recombination protein remains with the conditional replication shuttle vector; and wherein neither the IOBCV alone, nor the IOBCV in combination with the host cell can independently support homologous recombination; and    (b) growing the host cell under conditions in which the conditional replication shuttle vector cannot replicate, therein diluting out the conditional replication shuttle vector encoding the recombination protein, and thereby preventing further recombination events in the recombination deficient cells; and wherein the conditional replication shuttle vector cannot replicate in the host cell because the conditional replication shuttle vector comprises a R6K origin of replication and neither the host cell nor the BAC encode pir.    
     
     
         35 . The BAC of  claim 34  wherein the conditional replication shuttle vector further comprises two homologous nucleotide sequences that are homologous to each other but are not homologous to the BAC; wherein the two homologous nucleotide sequences are positioned on the conditional replication shuttle vector to be on opposite sides of the nucleic acid that selectively integrates into the particular nucleotide sequence; and wherein the resolution of the co-integrate is performed by a recombination event between the two homologous nucleotide sequences.  
     
     
         36 . A method of producing a non-human transgenic animal comprising: 
 (a) introducing a Bacterial or Bacteriophage-Derived Artificial Chromosome (BBPAC) into a eukaryotic cell; and    (b) placing the eukaryotic cell into a recipient animal, wherein the eukaryotic cell develops into the non-human transgenic animal;    wherein the BBPAC has been modified through homologous recombination in a RecA −  bacterial host cell that has been induced to support homologous recombination by the transient expression of a recombination protein; and wherein the eukaryotic cell is selected from the group consisting of a fertilized animal zygote and an embryonic stem cell.    
     
     
         37 . The method of  claim 36  wherein said eukaryotic cell is a fertilized animal zygote and said introducing is performed by pronuclear injecting the BBPAC into the fertilized animal zygote.  
     
     
         38 . The method of  claim 37  wherein the BBPAC is a Bacterial Artificial Chromosome (BAC); the animal is a mouse; and the fertilized animal zygote is a C57BL/6 mouse zygote.  
     
     
         39 . The method of  claim 38  wherein said eukaryotic cell is a mouse embryonic stem (ES) cell and said introducing is performed by transfecting the mouse ES cell.  
     
     
         40 . The method of  claim 39  wherein the BBPAC is a Bacterial Artificial Chromosome (BAC).  
     
     
         41 . The method of  claim 40  wherein the animal is a mammal.  
     
     
         42 . The method of  claim 41  wherein the mammal is a mouse.  
     
     
         43 . A method of producing a non-human transgenic animal comprising: 
 (a) introducing the BAC of  claim 34  into a eukaryotic cell; and    (b) placing the eukaryotic cell into a recipient animal, wherein the eukaryotic cell develops into the non-human transgenic animal.    
     
     
         44 . A method of producing a non-human transgenic animal comprising: 
 (a) introducing the BAC of  claim 35  into a eukaryotic cell; and    (b) placing the eukaryotic cell into a recipient animal, wherein the eukaryotic cell develops into the non-human transgenic animal.    
     
     
         45 . A non-human transgenic animal obtained by the method of  claim 44 .  
     
     
         46 . A non-human transgenic animal obtained by the method of  claim 43 .  
     
     
         47 . A non-human transgenic animal obtained by the method of  claim 36.