9. In an effort to produce gene knockout mice, a gene targeted homologous recombination was tried, with the exogenous. DNA containing neor gene (confer G-418 resistance) and tkHsv gene (confers sensitivity to the cytotoxic nucleotide analog ganciclovir). If the neor gene was inserted within the target gene in the exogenous DNA and considering that both homologous and non- homologous recombination (random integration) is taking place, which one of the following statements is NOT correct about the possible outcome of the experiment?
(1) Cells with non-homologous insertion will be sensitive to ganciclovir.
(2) Non-recombinant cells will be sensitive towards G-418 and resistant to ganciclovir.
(3) Homologous recombination will ensure that cells will be resistant to both ganciclovir and G-418.
(4) Homologous recombinants will grow in G-418 containing media but will be sensitive towards anciclovir.
Gene knockout mice are produced using a targeting vector where the neor gene (conferring G-418 resistance) disrupts the target gene between homologous arms, while tkHsv (conferring ganciclovir sensitivity) flanks outside these arms. Positive-negative selection enriches homologous recombinants: G-418 kills non-integrated cells, and ganciclovir eliminates random integrants retaining tkHsv. The incorrect statement is option 3, as homologous recombinants lack tkHsv and resist both drugs, unlike random events.
Vector Design and Recombination Types
The exogenous DNA integrates via homologous recombination (precise replacement at target locus) or non-homologous (random insertion). In homologous events, neor integrates within the locus, replacing target sequences and excluding tkHsv. Non-homologous events retain the full vector, including tkHsv outside homology arms. This setup ensures only desired knockouts survive dual selection.
Option-by-Option Analysis
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Option 1 (Correct): Non-homologous insertions retain tkHsv, producing thymidine kinase that sensitizes cells to ganciclovir, leading to death under negative selection.
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Option 2 (Correct): Non-recombinant cells (no integration) lack neor, dying in G-418; they retain endogenous machinery, resisting ganciclovir without tkHsv expression.
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Option 3 (Incorrect): Homologous recombination excludes tkHsv, granting resistance to both G-418 (via neor) and ganciclovir (no kinase produced); cells are not sensitive to either.
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Option 4 (Correct): Homologous recombinants express neor for G-418 growth but lack tkHsv, remaining insensitive (resistant) to ganciclovir.
Selection Strategy Benefits
Dual selection yields >1000-fold enrichment for homologous events over random ones, critical for embryonic stem cell screening before blastocyst injection. Random integrants survive G-418 but perish in ganciclovir, isolating true knockouts. This CSIR NET PYQ tests understanding of vector topology and marker placement.
