Direct cloning of isogenic murine DNA in yeast and relevance of isogenicity for targeting in embryonic stem cells.
Efficient gene targeting in embryonic stem cells requires that modifying DNA sequences are identical to those in the targeted chromosomal locus. Yet, there is a paucity of isogenic genomic clones for human cell lines and PCR amplification cannot be used in many mutation-sensitive applications. Here, we describe a novel method for the direct cloning of genomic DNA into a targeting vector, pRTVIR, using oligonucleotide-directed homologous recombination in yeast. We demonstrate the applicability of the method by constructing functional targeting vectors for mammalian genes Uhrf1 and Gfap. Whereas the isogenic targeting of the gene Uhrf1 showed a substantial increase in targeting efficiency compared to non-isogenic DNA in mouse E14 cells, E14-derived DNA performed better than the isogenic DNA in JM8 cells for both Uhrf1 and Gfap. Analysis of 70 C57BL/6-derived targeting vectors electroporated in JM8 and E14 cell lines in parallel showed a clear dependence on isogenicity for targeting, but for three genes isogenic DNA was found to be inhibitory. In summary, this study provides a straightforward methodological approach for the direct generation of isogenic gene targeting vectors.
Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden.
Claes
C
Andréasson
Anna J
AJ
Schick
Susanne M
SM
Pfeiffer
Mihail
M
Sarov
Francis
F
Stewart
Wolfgang
W
Wurst
Joel A
JA
Schick
D000818:Animals; D022762:CCAAT-Enhancer-Binding Proteins; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics*; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D018390:Gene Targeting; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D005904:Glial Fibrillary Acidic Protein; D059765:Homologous Recombination; D051379:Mice; D009419:Nerve Tissue Proteins / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics; D009928:Organ Specificity; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D044767:Ubiquitin-Protein Ligases
D022762:CCAAT-Enhancer-Binding Proteins; D005904:Glial Fibrillary Acidic Protein; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; C528900:glial fibrillary astrocytic protein, mouse; D004247:DNA; D044767:Ubiquitin-Protein Ligases; C117054:Uhrf1 protein, mouse