Practice question

Positive-negative selection enriches correctly targeted embryonic stem cells for knockout mouse production. The neomycin phosphotransferase gene placed inside homology arms confers G418 resistance, marking any vector integration event. Herpes simplex virus thymidine kinase located outside homology arms provides negative selection. Random integrants retain tk, which phosphorylates ganciclovir into toxic triphosphate nucleotides triggering cell death. Homologous double crossover excludes tk sequences, so true recombinants survive ganciclovir treatment while remaining G418 resistant, eliminating majority of non-specific insertions and ensuring isolation of correctly targeted clones.