10. A student while constructing knock-out mice isolated mouse embryonic, stem cells and introduced an engineered DNA into the cells. However, none of the mice were transgenic. On checking the cells containing DNA construct, he found that he had made a mistake in constructing the DNA since the cells were resistant to gancyclovir but sensitive to G418. Which one of the following constructs had he

The correct construct is option (3), in which both the neor and tk genes lie outside the homology arms so that random integration gives G418 resistance and ganciclovir sensitivity, while homologous recombination fails to insert the cassette and therefore produces no transgenic mice.

Concept behind the question

In gene targeting for knockout mice, neor acts as a positive selection marker because cells carrying an intact neor gene survive in G418, whereas tk (usually HSV‑tk) is a negative selection marker because cells expressing tk die in ganciclovir. Correctly designed vectors place neor inside the homology arms (so it is retained only after homologous recombination) and tk outside the homology arms (so it is lost after correct targeting but retained after random integration).

Because the ES cells in the question are resistant to ganciclovir but sensitive to G418, they must have lost tk but do not contain a functional neor cassette, meaning that successful recombinants (if any) cannot be selected with G418 and hence no transgenic mice appear. This phenotype matches a vector in which neor lies outside the homology arms together with tk so that homologous recombination removes both markers from the targeted locus, whereas random integrants are doubly resistant (G418R, GANCs).

Option‑wise explanation

Option (1)

In this construct neor is located inside the homology arms and tk outside. On random integration, cells receive both neor and tk and are therefore G418 resistant and ganciclovir sensitive. On homologous recombination, the recombined allele retains neor but loses tk, producing G418‑resistant and ganciclovir‑resistant targeted clones that can generate transgenic mice. Thus option (1) would work correctly and cannot explain the failure described.

Option (2)

Here neor lies just outside the homology arm, whereas tk sits within the region of homology. Random integrants again express both neor and tk and are killed by ganciclovir but survive G418, so they can be eliminated by negative selection. Correct homologous recombination inserts tk into the locus and removes neor; such cells are G418 sensitive but ganciclovir sensitive as well, which does not match the observed ganciclovir resistance. Hence option (2) is inconsistent with the phenotype.

Option (3) – Correct

In this vector both neor and tk lie outside the homology arms. Random integration brings both markers into the genome so those cells are G418 resistant and ganciclovir sensitive and are removed by ganciclovir treatment. Homologous recombination, however, uses only the homology arms and excludes the external neor‑tk cassette; therefore correctly targeted cells lack both genes and are sensitive to both drugs, so they cannot be selected and will not yield identifiable transgenic mice, exactly as in the question (ganciclovir‑resistant colonies are merely those with degraded or non‑expressed tk outside the target site).

Option (4)

Option (4) depicts a fused neor‑tk cassette inside the homology arms so that both markers insert into the locus on homologous recombination. Targeted cells would then be simultaneously G418 resistant and ganciclovir sensitive, whereas random integrants might disrupt the cassette variably but would still often express one or both markers; this pattern again does not match the observed ganciclovir resistance and G418 sensitivity.

Short SEO‑style introduction (using the key phrase):
Knockout mice neor tk ganciclovir G418 selection strategies rely on precise placement of positive and negative markers within targeting constructs. When neor and tk cassettes are arranged incorrectly, ES cells show unexpected drug‑resistance patterns and fail to generate transgenic animals, as illustrated by this classical gene‑targeting problem.