Targeting the Rosa26 gene in human embryonic stem cells
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 embryonic stem cell, which method comprises introducing exogenous DNA into the human Rosa26 gene in an isolated human embryonic stem cell.
2. The method of claim 1 , wherein the exogenous DNA is introduced into the human embryonic stem cell by homologous recombination with a targeting vector comprising said exogenous DNA flanked by DNA sequences homologous to the human Rosa26 gene.
3. The method of claim 2 , wherein the targeting vector further comprises functional sequences selected from the group consisting of tags for protein detection, enhancers, selection markers, and combinations thereof.
4. The method of claim 2 , wherein the DNA sequences homologous to the human Rosa26 gene are derived from the 5′ and 3′ flanking arm of the human Rosa26 gene.
5. The method of claim 1 , wherein the human embryonic stem cell is selected from the group consisting of primary cells and immortalized cells.
6. The method of claim 1 , wherein the gene of interest is selected from the group consisting of a gene encoding a recombinase, a reporter and mutations and combinations thereof.
7. The method of claim 1 , wherein the heterologous 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.
8. The method of claim 1 wherein the gene expression cassette further comprises one or more additional sequences selected from the group consisting of marker genes, one or more recombinase recognition sites which may be the same or different, poly A introns, and combinations thereof.
9. The method of claim 1 , wherein the expression cassette comprises one or more functional sequences selected from the group consisting of a viral splice acceptor, a loxP-flanked promoterless neomycin resistance gene, an inverted REP variant, loxP2272 sites, and combinations thereof.
10. The method of claim 1 , wherein the expression cassette comprises the following elements in sequential order: (a) a viral splice acceptor, (b) loxP, (c) a promoterless neomycin resistance gene, (d) loxP2272, (e) an inverted tdRFP, (f) loxP, and (g) loxP2272.
11. The method of claim 10 wherein said expression cassette comprises a DNA sequence coding for a Cre recombinase and loxP and loxP2272 sites positioned such that following expression of Cre recombinase, the neomycin resistance gene is removed and the tdRFP inverted, placing it under control of the endogenous human Rosa26 promoter.
12. The method of claim 1 wherein said exogenous DNA is a gene expression cassette comprising a gene of interest operatively linked to a heterologous promoter.
13. The method of claim 1 wherein said exogenous DNA is a gene expression cassette comprising a gene of interest, and wherein the exogenous DNA is introduced into the Rosa26 gene by homologous recombination, such that expression of the DNA sequence is under the control of the endogenous Rosa26 promoter.