Stem cell gene targeting
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. A method for modifying a human Rosa26 gene in an isolated human stem cell, which method comprises introducing an expression cassette comprising a nucleic acid sequence encoding a protein, such that said nucleic acid sequence is operatively linked to a promoter heterologous to the human Rosa26 gene, or the endogenous human Rosa26 promoter.
2. The method of claim 1 , wherein the expression cassette is introduced into the human stem cell by homologous recombination with a targeting vector comprising said expression cassette flanked by DNA sequences homologous to the human Rosa26 gene.
3. The method of claim 1 , wherein the human stem cell is a primary cell or an immortalized cell.
4. The method of claim 1 , wherein the nucleic acid sequence encodes a recombinase, or a reporter.
5. The method of claim 1 , wherein the promoter heterologous to the human Rosa26 gene 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.
6. The method of claim 1 wherein the expression cassette further comprises a marker gene, one or more recombinase recognition site, a poly A signal, an intron, or combinations thereof.
7. The method of claim 1 , wherein the expression cassette comprises a viral splice acceptor, a loxP-flanked promoterless neomycin resistance gene, an inverted RFP variant, loxP2272 sites, or combinations thereof.
8. The method of claim 1 , 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.
9. The method of claim 2 , wherein the targeting vector further comprises tags for protein detection, enhancers, selection markers, or combinations thereof.
10. The method of claim 1 , wherein said promoter is heterologous to the human Rosa26 gene.
11. The method of claim 1 wherein said promoter is the endogenous Rosa26 promoter.