Practice question

Neomycin resistance gene neo confers survival in G418 or Geneticin through aminoglycoside phosphotransferase inactivating the drug. HSV-tk alone cannot provide G418 protection; it only sensitizes cells to ganciclovir via toxic metabolite formation. A vector lacking neo but retaining tk outside homology regions possesses no positive selection marker, so electroporated embryonic stem cells cannot detoxify G418 effectively. Even if random integration inserts tk, cells remain sensitive to G418 and die during selection. Therefore loss of positive marker aborts enrichment of targeted clones altogether preventing isolation.