Methods of modifying genes in eukaryotic cells

Abstract

Methods of creating mutations in genomic exons by inserting introns into the genomic exons via homologous recombination. Also, methods are provided for introducing modifications into genomic exons by inserting introns into the genomic exons via homologous recombination such that a mature mRNA transcript produced from a genomic region of the genome comprising the genomic exon does not contain the modification are provided. The methods provide for a rapid method for introducing mutations and/or modifications of any type into a mammalian cell genome.

1 . A method creating a mutation in a genomic exon in the genome of a mammalian embryonic stem (ES) cell, comprising: 
 (a) introducing an intron into the genomic exon by homologous recombination such that a mutation is created in the genomic exon, wherein the intron comprises at least two site-specific recombination sites oriented in the same direction with respect to one another;    (b) introducing the embryonic stem cell comprising the mutated genomic exon into a blastocyst; and    (c) introducing the blastocyst of (b) into a surrogate mother for gestation of offspring having the mutated genomic exon.    
   
   
       2 . The method of  claim 1 , wherein the site-specific recombination sites are selected from the group comprising loxP and FRT sites.  
   
   
       3 . The method of  claim 1 , further comprising exposing the offspring having the mutated genomic exon to a recombinase that recognizes the site-specific recombination sites such that the DNA sequence between the site-specific recombination sites is excised.  
   
   
       4 . The method of  claim 1 , further comprising exposing the offspring of step (c) to a recombinase that recognizes the site-specific recombination sites such that the sequence between the site-specific recombination sites is excised.  
   
   
       5 . A method introducing a modification into a genomic exon in the genome of a eukaryotic cell, comprising introducing an intron into a genomic exon by homologous recombination such that a mature mRNA transcript produced from a genomic region of the genome comprising the genomic exon does not contain the modification, wherein the method optionally comprises at least one of: (i) employing an intron that comprises at least one site-specific recombination site, and (ii) introducing a site-specific recombination site into a region outside of the genomic exon by homologous recombination.  
   
   
       6 . The method of  claim 5 , wherein the intron comprises at least one site-specific recombination site, and wherein the at least one site-specific recombination site in the intron and the site-specific recombination site in the region outside the genomic exon are in one of the following orientations: 
 (i) in the same direction with respect to one another such that excision of the DNA sequence between them occurs as a result of site-specific recombination; or    (ii) in the opposite direction with respect to one another to allow for inversion of the DNA sequence between them following site-specific recombination.    
   
   
       7 . The method of  claim 6 , further comprising introducing an embryonic stem cell comprising the genomic exon containing the modification into a blastocyst, and then introducing the blastocyst into a surrogate mother for gestation of offspring having the modified genomic exon.  
   
   
       8 . The method of  claim 7 , wherein 
 (a) the site-specific recombination sites are oriented in the same direction, and the method further comprises exposing the offspring having the modified genomic exon to a recombinase that recognizes the site-specific recombination sites such that the sequence between the site-specific recombination sites is excised; or    (b) the site-specific recombination sites are oriented in opposite directions, and the method further comprises exposing the offspring having the modified genomic exon to a recombinase that recognizes the site-specific recombination sites such that the sequence between the site-specific recombination sites is inverted.    
   
   
       9 . The method of  claim 5 , wherein the intron comprises at least one site-specific recombination site, and wherein a site-specific recombination site is introduced into a region outside of the genomic exon by homologous recombination, wherein the site-specific recombination site in the intron and the site-specific recombination site in the region outside the genomic exon are oriented in the same direction with respect to one another such that excision of the DNA sequence between them occurs as a result of site-specific recombination, and wherein the eukaryotic cell is an embryonic stem cell, and the embyronic stem cell comprising the genomic exon containing the modification is introduced into a blastocyst and then the blastocyst is introduced into a surrogate mother for gestation of offspring having the modified genomic exon, and the method optionally further comprises a step of exposing offspring having the modified genomic exon to a recombinase that recognizes the site-specific recombination sites such that the sequence between the site-specific recombination sites is excised.  
   
   
       10 . A method introducing a modification into a genomic exon in the genome of an embryonic stem (ES) cell, comprising: 
 (a) introducing an intron into a genomic exon by homologous recombination such that a mature mRNA transcript produced from a genomic region of the genome comprising the genomic exon does not contain the modification, wherein the intron comprises at least one site-specific recombination site;    (b) introducing a site specific recombination site into a region outside of the genomic exon by homologous recombination, wherein the site-specific recombination site in the intron and the site-specific recombination site in the region outside the genomic exon are oriented in the opposite direction with respect to one another to allow for inversion of the DNA sequence between them following site-specific recombination;    (c) introducing an embryonic stem cell comprising the genomic exon containing the modification into a blastocyst; and    (d) introducing the blastocyst of (c) into a surrogate mother for gestation of offspring having the modified genomic exon.    
   
   
       11 . The method of  claim 9 , further comprising: 
 (e) exposing the offspring of step (d) to a recombinase that recognizes the site-specific recombination sites such that the sequence between the site-specific recombination sites is inverted.    
   
   
       12 . A method according to  claim 1 , wherein the mutation is a transcription termination sequence, the mutation is introduced into an antisense strand of a genomic exon in a mammalian cell, and the transcription termination sequence is oriented in the antisense direction with respect to the genomic exon into which it is to be introduced.  
   
   
       13 . The method of  claim 12 , wherein the intron further comprises at least two site-specific recombination sites, wherein the site-specific recombination sites flank the transcriptional termination signal sequence.  
   
   
       14 . The method of  claim 12 , wherein the site-specific recombination sites are oriented in the opposite direction with respect to one another such that inversion of the transcriptional termination signal sequence occurs as a result of site-specific recombination.