Your student is trying to create a "Gene X" knockout mouse by introducing a recombinant construct into embryonic stem (ES) cells, selecting transformants, and introducing the transformed cells into mouse blastocysts

Question

He designed his construct as follows: NeoR----region of homology to Gene X---HSV tk--- region of homology to Gene X where NeoR is the neomycin-resistance gene and HSV tk is the Herpes Simplex virus thymidine kinase gene. He transforms the ES cells and exposes them to G418 and ganciclovir. Explain to your student he failed to recover transformed ES cells.

Answer 1

If his construct incorporated into the mouse DNA by illegitimate recombination, both the neomycin resistance gene and the thymidine kinase gene would be incorporated into the genome. These cells would be resistant to neomycin, but sensitive to ganciclovir, and would die.
If the construct incorporated into the mouse DNA by homologous recombination, only the gene flanked by the regions homologous to Gene X would be incorporated. In your student's construct, this gene was the tk gene. The neomycin resistance gene, outside the regions of homology, would not be incorporated. These cells would be sensitive to both G418 and ganciclovir, and would die.