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Somatic cell and molecular genetics

Targeted gene replacement at the endogenous APRT locus in CHO cells.


PMID 2237639

Abstract

We demonstrate the feasibility of targeted gene replacement at an endogenous, chromosomal gene locus in cultured mammalian cells, employing a two-step strategy similar to an approach routinely used for genetic manipulation in yeast. Utilizing an APRT+ recombinant generated by targeted integration of plasmid sequences (including a functional copy of the gpt gene) at the CHO APRT locus, we have been able to select gpt- "pop-out" recombinants that have arisen by intrachromosomal recombination between APRT direct repeats at the targeted integration site. Reciprocal exchanges leading to "pop-out" of integrated plasmid/gpt gene sequences occur at a rate of approximately 6.3 x 10(-6) per cell generation. Depending on the site of crossover, such "pop-out" events result in either replacement or restoration of the original APRT target gene sequence.