Gene Replacement - The technique of positive negative selection permits the recovery of such ES cell lines in which homologous recombination leads to the disruption of the gene of interest. In contrast, the 'hit-and-run method' was designed to recover those ES cells in which a defective endogenous gene is replaced by homologous recombination with a functional gene, or a normal endogenous gene is replaced with a gene modified by site directed mutagenesis.

This method is also known as the 'in-out' strategy since the vector sequences are first integrated into the genome, and then they are removed out of the genome.

In this method, the gene of interest is not disrupted by the neo gene; the neo and HSV- Tk genes are integrated in the vector alongside the gene of interest.