Enhanced selection for homologous-recombinant embryonic stem cell clones with a neomycin phosphotransferase gene in...

Gene targeting in embryonic stem cells via homologous recombination can occur at a very low frequency. In order to enrich the selection for homologous recombinants, replacement targeting vectors are now commonly used that contain the thymidine kinase gene placed outside of the targeted homology. The additional negative selection requires the presence of antiviral drugs in the culture medium which are known to reduce the ability of embryonic stem cells to colonize the germ line. We have therefore tested alternative negative selection procedures with replacement targeting vectors that allow the expression of either a ribozyme directed against the neomycin-resistance gene (neo(r)) or of an antisense neo(r) RNA at random integration sites. The hammerhead ribozyme was found to be catalytically inactive in embryonic stem cell cultures maintained in the presence of the selecting drug neomycin. Thus, the replacement targeting vector that contains ribozyme sequences did not enhance the frequency of homologous recombination. However, placing a promotor sequence that can enable the transcription of antisense neo(r) RNA outside of the targeted homology led to a significant enrichment of the selection for homologous recombination. This enrichment is similar to previously reported enrichments obtained with the thymidine kinase gene. The advantage, however, is that no antiviral drugs are needed for the selection.