Gene targeting in mice: Knockout mice (loss-of-function for a chosen gene) are most commonly generated by which approach?

Introduction:
Knockout mice enable precise functional studies by disrupting specific genes. The classic pre-CRISPR approach uses homologous recombination in embryonic stem (ES) cells to replace or disrupt the target locus. This question asks you to pick the method that defines traditional knockout generation.

Given Data / Assumptions:

• ES cells can contribute to the germline when introduced into blastocysts.
• Gene targeting constructs contain homology arms flanking a selectable cassette.
• Chimeric founders transmit the targeted allele to progeny.

Concept / Approach:
Transfect ES cells with a targeting vector designed for homologous recombination. Select for correctly targeted clones using positive/negative selection (e.g., neomycin resistance and ganciclovir counterselection). Inject targeted ES cells into blastocysts to create chimeric mice, breed to germline transmission, and intercross to obtain homozygous knockouts. Although CRISPR/Cas systems now streamline this, the canonical method remains ES cell gene targeting for many historical and some current applications.

Step-by-Step Solution:

Verification / Alternative check:
Genotyping by Southern blot or PCR across junctions confirms proper homologous recombination; Mendelian inheritance in offspring validates germline transmission of the knockout allele.

Why Other Options Are Wrong:

• Random mutagenesis or retroviral insertion lacks locus specificity.
• Cell fusion chimeras without targeting do not guarantee gene disruption.
• Somatic nuclear transfer is cloning, not gene targeting per se.

Common Pitfalls:
Confusing general chimera creation with targeted gene disruption; chimerism is a step in ES-based knockouts but targeting is the defining feature.

Final Answer:
Transfecting embryonic stem (ES) cells with an engineered gene construct and selecting homologous recombinants