Methods of modifying eukaryotic cells

Abstract

A method for engineering and utilizing large DNA vectors to target, via homologous recombination, and modify, in any desirable fashion, endogenous genes and chromosomal loci in eukaryotic cells. These large DNA targeting vectors for eukaryotic cells, termed LTVECs, are derived from fragments of cloned genomic DNA larger than those typically used by other approaches intended to perform homologous targeting in eukaryotic cells. Also provided is a rapid and convenient method of detecting eukaryotic cells in which the LTVEC has correctly targeted and modified the desired endogenous genes(s) or chromosomal locus (loci) as well as the use of these cells to generate organisms bearing the genetic modification.

1 . A method for genetically modifying an endogenous gene or chromosomal locus of interest in eukaryotic cells, comprising: 
 a) obtaining a large cloned genomic fragment containing a DNA sequence of interest;    b) using bacterial homologous recombination to genetically modify the large cloned genomic fragment of (a) to create a large targeting vector for use in the eukaryotic cells (LTVEC);    c) introducing the LTVEC of (b) into the eukaryotic cells to modify the endogenous gene or chromosomal locus in the cells; and    d) using a quantitative assay to detect modification of allele (MOA) in the eukaryotic cells of (c) to identify those eukaryotic cells in which the endogenous gene or chromosomal locus has been genetically modified.    
     
     
         2 . The method of  claim 1  wherein the large cloned genomic fragment containing a DNA sequence is homologous to the endogenous gene or chromosomal locus of interest.  
     
     
         3 . The method of  claim 1  wherein the genetic modification to the endogenous gene or chromosomal locus comprises deletion of a coding sequence, gene segment, or regulatory element; alteration of a coding sequence, gene segment, or regulatory element; insertion of a new coding sequence, gene segment, or regulatory element; creation of a conditional allele; or replacement of a coding sequence or gene segment from one species with an homologous or orthologous coding sequence from the same or a different species.  
     
     
         4 . The method of  claim 3  wherein the alteration of a coding sequence, gene segment, or regulatory element comprises a substitution, addition, or fusion.  
     
     
         5 . The method of  claim 4  wherein the fusion comprises an epitope tag or bifunctional protein.  
     
     
         6 . The method of  claim 1  wherein the quantitative assay comprises quantitative PCR, FISH, comparative genomic hybridization, isothermic DNA amplification, quantitative hybridization to an immobilized probe, Invader Probes®, or MMP assays®.  
     
     
         7 . The method of  claim 6  wherein the quantitative PCR comprises TaqMan®, Molecular Beacon, or Eclipse™ probe technology.  
     
     
         8 . The method of  claim 1  wherein the eukaryotic cell is a mammalian embryonic stem cell.  
     
     
         9 . The method of  claim 8  wherein the embryonic stem cell is a mouse, rat, or other rodent embryonic stem cell.  
     
     
         10 . The method of  claim 1  wherein the endogenous gene or chromosomal locus is a mammalian gene or chromosomal locus.  
     
     
         11 . The method of  claim 10  wherein the endogenous gene or chromosomal locus is a human gene or chromosomal locus.  
     
     
         12 . The method of  claim 10  wherein the endogenous gene or chromosomal locus is a mouse, rat, or other rodent gene or chromosomal locus.  
     
     
         13 . The method of  claim 1  wherein the LTVEC is capable of accommodating large DNA fragments greater than 20 kb.  
     
     
         14 . The method of  claim 13  wherein the LTVEC is capable of accommodating large DNA fragments greater than 100 kb.  
     
     
         15 . A method for genetically modifying an endogenous gene or chromosomal locus of interest in mouse embryonic stem cells, comprising: 
 a) obtaining a large cloned genomic fragment greater than 20 kb which contains a DNA sequence of interest, wherein the large cloned DNA fragment is homologous to the endogenous gene or chromosomal locus;    b) using bacterial homologous recombination to genetically modify the large cloned genomic fragment of (a) to create a large targeting vector for use in the mouse embryonic stem cells, wherein the genetic modification is deletion of a coding sequence, gene segment, or regulatory element;    c) introducing the large targeting vector of (b) into the mouse embryonic stem cells to modify the endogenous gene or chromosomal locus in the cells; and    d) using a quantitative assay to detect modification of allele (MOA) in the mouse embryonic stem cells of (c) to identify those mouse embryonic stem cells in which the endogenous gene or chromosomal locus has been genetically modified, wherein the quantitative assay is quantitative PCR.    
     
     
         16 . A genetically modified endogenous gene or chromosomal locus produced by the method of any one of claims  1  or  15 .  
     
     
         17 . The genetically modified endogenous gene or chromosomal locus of  claim 16  wherein the genetic modification to the endogenous gene or chromosomal locus comprises deletion of a coding sequence, gene segment, or regulatory element; alteration of a coding sequence, gene segment, or regulatory element; insertion of a new coding sequence, gene segment, or regulatory element; creation of a conditional allele; or replacement of a coding sequence or gene segment from one species with an homologous or orthologous coding sequence from the same or a different species.  
     
     
         18 . The genetically modified endogenous gene or chromosomal locus of  claim 17  wherein the alteration of a coding sequence, gene segment, or regulatory element comprises a substitution, addition, or fusion.  
     
     
         19 . The genetically modified endogenous gene or chromosomal locus of  claim 18  wherein the fusion comprises an epitope tag or bifunctional protein.  
     
     
         20 . A genetically modified eukaryotic cell produced by the method of  claim 1 .  
     
     
         21 . A genetically modified mouse embryonic stem cell produced by the method of  claim 15 .  
     
     
         22 . The genetically modified eukaryotic cell of  claim 20  wherein the genetic modification to the endogenous gene or chromosomal locus comprises deletion of a coding sequence, gene segment, or regulatory element; alteration of a coding sequence, gene segment, or regulatory element; insertion of a new coding sequence, gene segment, or regulatory element; creation of a conditional allele; or replacement of a coding sequence or gene segment from one species with an homologous or orthologous coding sequence from the same or a different species.  
     
     
         23 . The genetically modified eukaryotic cell of  claim 22  wherein the alteration of a coding sequence, gene segment, or regulatory element comprises a substitution, addition, or fusion.  
     
     
         24 . The genetically modified eukaryotic cell of  claim 23  wherein the fusion comprises an epitope tag or bifunctional protein.  
     
     
         25 . A non-human organism containing a genetically modified endogenous gene or chromosomal locus produced by the method of  claim 1 .  
     
     
         26 . A mouse containing a genetically modified endogenous gene or chromosomal locus produced by the method of  claim 15 .  
     
     
         27 . A non-human organism produced from the genetically modified eukaryotic cell of any one of claims  20 ,  22 ,  23 , or  24 .  
     
     
         28 . A mouse produced from the genetically modified mouse embryonic stem cell of  claim 21 .  
     
     
         29 . A genetically modified embryonic stem cell produced by the method of  claim 1 .  
     
     
         30 . The genetically modified embryonic stem cell of  claim 29  wherein the genetic modification to the endogenous gene or chromosomal locus comprises deletion of a coding sequence, gene segment, or regulatory element; alteration of a coding sequence, gene segment, or regulatory element; insertion of a new coding sequence, gene segment, or regulatory element; creation of a conditional allele; or replacement of a coding sequence or gene segment from one species with an homologous or orthologous coding sequence from a different species.  
     
     
         31 . The genetically modified embryonic stem cell of  claim 30  wherein the alteration of a coding sequence, gene segment, or regulatory element comprises a substitution, addition, or fusion.  
     
     
         32 . The genetically modified embryonic stem cell of  claim 31  wherein the fusion comprises an epitope tag or bifunctional protein.  
     
     
         33 . A non-human organism containing a genetically modified endogenous gene or chromosomal locus of interest, produced by a method comprising the steps of: 
 a) obtaining a large cloned genomic fragment containing a DNA sequence of interest;    b) using bacterial homologous recombination to genetically modify the large cloned genomic fragment of (a) to create a large targeting vector (LTVEC) for use in embryonic stem cells;    c) introducing the LTVEC of (b) into the embryonic stem cells to modify the endogenous gene or chromosomal locus in the cells;    d) using a quantitative assay to detect modification of allele (MOA) in the embryonic stem cells of (c) to identify those embryonic stem cells in which the endogenous gene or chromosomal locus has been genetically modified;    e) introducing the embryonic stem cell of (d) into a blastocyst; and    f) introducing the blastocyst of (e) into a surrogate mother for gestation.    
     
     
         34 . A mouse containing a genetically modified endogenous gene or chromosomal locus of interest, produced by a method comprising the steps of: 
 a) obtaining a large cloned genomic fragment greater than 20 kb which contains a DNA sequence of interest, wherein the large cloned DNA fragment is homologous to the endogenous gene or chromosomal locus;    b) using bacterial homologous recombination to genetically modify the large cloned genomic fragment of (a) to create a large targeting vector for use in the mouse embryonic stem cells, wherein the genetic modification is deletion of a coding sequence, gene segment, or regulatory element;    c) introducing the large targeting vector of (b) into the mouse embryonic stem cells to modify the endogenous gene or chromosomal locus in the cells; and    d) using a quantitative assay to detect modification of allele (MOA) in the mouse embryonic stem cells of (c) to identify those mouse embryonic stem cells in which the endogenous gene or chromosomal locus has been genetically modified, wherein the quantitative assay is quantitative PCR;    e) introducing the mouse embryonic stem cell of (d) into a blastocyst; and    f) introducing the blastocyst of (e) into a surrogate mother for gestation.    
     
     
         35 . A non-human organism containing a genetically modified endogenous gene or chromosomal locus, produced by a method comprising the steps of: 
 a) obtaining a large cloned genomic fragment containing a DNA sequence of interest;    b) using bacterial homologous recombination to genetically modify the large cloned genomic fragment of (a) to create a large targeting vector for use in eukaryotic cells (LTVEC);    c) introducing the LTVEC of (b) into the eukaryotic cells to genetically modify the endogenous gene or chromosomal locus in the cells;    d) using a quantitative assay to detect modification of allele (MOA) in the eukaryotic cells of (c) to identify those eukaryotic cells in which the endogenous gene or chromosomal locus has been genetically modified;    e) removing the nucleus from the eukaryotic cell of (d);    f) introducing the nucleus of (e) into an oocyte; and    g) introducing the oocyte of (f) into a surrogate mother for gestation.    
     
     
         36 . A non-human organism containing a genetically modified endogenous gene or chromosomal locus, produced by a method comprising the steps of: 
 a) obtaining a large cloned genomic fragment containing a DNA sequence of interest;    b) using bacterial homologous recombination to genetically modify the large cloned genomic fragment of (a) to create a large targeting vector for use in eukaryotic cells (LTVEC);    c) introducing the LTVEC of (b) into the eukaryotic cells to genetically modify the endogenous gene or chromosomal locus in the cells;    d) using a quantitative assay to detect modification of allele (MOA) in the eukaryotic cells of (c) to identify those eukaryotic cells in which the endogenous gene or chromosomal locus has been genetically modified;    e) fusing the eukaryotic cell of (d) with another eukaryotic cell; and    f) introducing the fused eukaryotic cell of (e) into a surrogate mother for gestation.    
     
     
         37 . The non-human organism of any one of claims  33 ,  35 , or  36  wherein the large cloned genomic fragment containing a DNA sequence is homologous to the endogenous gene or chromosomal locus of interest.  
     
     
         38 . The non-human organism of any one of claims  25 ,  33 ,  35 , or  36  wherein the non-human organism is a mouse, rat, or other rodent.  
     
     
         39 . The non-human organism of  claim 33  wherein the blastocyst is a mouse, rat, or other rodent blastocyst.  
     
     
         40 . The non-human organism of  claim 35  wherein the oocyte is a mouse, rat, or other rodent oocyte.  
     
     
         41 . The non-human organism of any one of claims  33 ,  35 , or  36  wherein the surrogate mother is a mouse, rat, or other rodent.  
     
     
         42 . The non-human organism of  claim 33  wherein the embryonic stem cell is a mammalian embryonic stem cell.  
     
     
         43 . The non-human organism of  claim 42  wherein the mammalian embryonic stem cell is a mouse, rat, or other rodent embryonic stem cell.  
     
     
         44 . The non-human organism of any one of claims  33 ,  35 , or  36  wherein the genetic modification to the endogenous gene or chromosomal locus comprises deletion of a coding sequence, gene segment, or regulatory element; alteration of a coding sequence, gene segment, or regulatory element; insertion of a new coding sequence, gene segment, or regulatory element; creation of a conditional allele; or replacement of a coding sequence or gene segment from one species with an homologous or orthologous coding sequence from the same or a different species.  
     
     
         45 . The non-human-organism of  claim 44  wherein the alteration of a coding sequence, gene segment, or regulatory element comprises a substitution, addition, or fusion.  
     
     
         46 . The non-human organism of  claim 45  wherein the fusion comprises an epitope tag or bifunctional protein.  
     
     
         47 . The non-human organism of any one of claims  33 ,  35 , or  36  wherein the quantitative assay comprises quantitative PCR, FISH, comparative genomic hybridization, isothermic DNA amplification, quantitative hybridization to an immobilized probe, Invader Probes®, or MMP® assays.  
     
     
         48 . The non-human organism of  claim 47  wherein the quantitative PCR comprises TaqMan®, Molecular Beacon, or Eclipse™ probe technology.s.  
     
     
         49 . The use of the genetically modified eukaryotic cell of  claim 20  for the production of a non-human organism.  
     
     
         50 . The use of the genetically modified mouse embryonic stem cell of  claim 21  for the production of a mouse.  
     
     
         51 . The use of the genetically modified embryonic stem cell of  claim 29  for the production of a non-human organism.  
     
     
         52 . The method of claims  1  or  15 , wherein about 1-5 μg of the large targeting vector of (c) is introduced to about 1×10 7  cells.  
     
     
         53 . The mouse of  claim 34 , wherein about 1-5 μg of the large targeting vector of (c) is introduced to about 1×10 7  cells.  
     
     
         54 . The non-human organism of claims  33 ,  35 , or  36 , wherein about 1-5 μg of the large targeting vector of (c) is introduced to about 1×10 7  cells.