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

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.

Spontaneous CHO APRT heterozygotes reflect high-frequency, allele-specific deletion of the chromosome Z4 APRT gene.

In Chinese hamster ovary (CHO) cells, heterozygotes for the adenine phosphoribosyltransferase (APRT) locus arise spontaneously at high frequencies. Paradoxically, such heterozygotes yield APRT mutants only at much lower spontaneous rates, suggesting that the high-frequency event may occur at only one of the two APRT genes. In an attempt to understand the genetic basis for the apparent refractivity of one of the APRT alleles to the high-frequency genetic event and to determine whether differences in the genomic environments of the two CHO APRT alleles specifically render one gene more susceptible to high-frequency spontaneous deletion or inactivation, we have mapped the wild-type APRT allele in 16 independently derived spontaneous APRT heterozygotes. In 15 of these 16 heterozygotes, the functional, wild-type APRT gene was found to reside on the Z7 chromosome, indicating that the high-frequency event is indeed highly specific for the Z4 APRT allele. All but one of these heterozygotes were hemizygous for the APRT locus, suggesting that the high-frequency event generally involves deletion rather than spontaneous inactivation or mutation of the Z4 APRT allele.

Preferential loss or inactivation of chromosome Z4 APRT allele in CHO cells.

In CHO cells, heterozygotes for the adenine phosphoribosyltransferase (APRT) locus arise spontaneously at high frequencies. However, such heterozygotes always yield APRT- mutants at low spontaneous rates. In an attempt to determine whether differences in the genomic environments of the two CHO APRT alleles might render one gene more susceptible to high-frequency spontaneous inactivation or deletion, we have mapped the functional APRT allele in four different spontaneous APRT heterozygotes. In each case, the functional APRT gene was found to reside on the Z7 chromosome; it was always the Z4 APRT allele that had been lost or inactivated. Two of these heterozygotes were shown to be physically hemizygous while the other two retained two copies of the APRT gene, indicating that the high-frequency event can involve either spontaneous deletion or inactivation.

Targeted homologous recombination at the endogenous adenine phosphoribosyltransferase locus in Chinese hamster cells.

We have developed a system that permits analysis of targeted homologous recombination at an endogenous, chromosomal gene locus in cultured mammalian cells. Using a hemizygous, adenine phosphoribosyltransferase (APRT)-deficient, Chinese hamster ovary (CHO) cell mutant as a transfection recipient, we have demonstrated correction of a nonrevertible deletion mutation by targeted homologous recombination. Transfection with a plasmid carrying a fragment of the APRT gene yielded APRT+ recombinants at a frequency of approximately 4.1 x 10(-7). The ratio of targeted recombination to nontargeted integrations of plasmid sequences was approximately 1:4000. Analysis of 31 independent APRT+ recombinants revealed conversions of the endogenous APRT gene, targeted integration at the APRT locus, and a third class of events in which the plasmid donor APRT fragment was converted to a full-length, functional gene.