Stem cell gene targeting

Abstract

The invention provides a method for generating a transgenic eukaryotic cell population having a modified human Rosa26 locus, which method includes introducing a functional DNA sequence into the human Rosa26 locus of starting eukaryotic cells. Also provided are targeting vectors useful in the method, as well as a cell population and a transgenic non-human animal comprising a modified human Rosa26 locus. Finally, the invention provides an isolated DNA sequence corresponding to the human Rosa26 locus.

1 - 14 . (canceled) 
     
     
         15 . A targeting vector comprising an expression cassette comprising a nucleic acid encoding a protein, said expression cassette flanked by DNA sequences homologous to the human Rosa26 gene. 
     
     
         16 . An ex vivo human cell population comprising an exogenous nucleic acid in the human Rosa26 gene. 
     
     
         17 .- 20 . (canceled) 
     
     
         21 . The targeting vector of  claim 15  wherein the expression cassette further comprises a promoter operably linked to the nucleic acid encoding the protein. 
     
     
         22 . The targeting vector of  claim 21  wherein the promoter is selected from the group consisting of a constitutive ubiquitous promoter, a constitutive tissue specific promoter, an inducible ubiquitous promoter and an inducible tissue specific promoter. 
     
     
         23 . The targeting vector of  claim 21  wherein the promoter is heterologous to the human Rosa26 gene. 
     
     
         24 . The targeting vector of  claim 21  wherein the promoter is the endogenous human Rosa26 promoter. 
     
     
         25 . The targeting vector of  claim 15  wherein the sequences homologous to the human Rosa26 gene are derived from the 5′ and 3′ flanking arms of the human Rosa26 gene. 
     
     
         26 . The targeting vector of  claim 15  further comprising tags for protein detection, enhancers, selection markers, and combinations thereof. 
     
     
         27 . The targeting vector of  claim 15  wherein the nucleic acid encodes a recombinase or a reporter. 
     
     
         28 . The targeting vector of  claim 15  wherein the expression cassette further comprises a marker gene, one or more recombinase recognition sites, a poly A signal, an intron, or combinations thereof. 
     
     
         29 . The targeting vector of  claim 15  wherein the expression cassette further comprises a viral splice acceptor, a loxP-flanked promoterless neomycin resistance gene, an inverted RFP variant, loxP2272 sites, or combinations thereof. 
     
     
         30 . The targeting vector of  claim 15  wherein the expression cassette comprises the following elements in sequential order: (a) a viral splice acceptor, (b) a loxP site, (c) a promoterless neomycin resistance gene, (d) a loxP2272 site, (e) an inverted nucleic acid sequence encoding the protein, (f) a loxP site, and (g) a loxP2272 site.