The Mechanism of Mammalian Gene Replacement Is Consistent with the Formation of Long Regions of Heteroduplex DNA Associated with Two Crossing-Over Events

ABSTRACT In this study, the mechanism of mammalian gene replacement was investigated. The system is based on detecting homologous recombination between transferred vector DNA and the haploid, chromosomal immunoglobulin μ-δ region in a murine hybridoma cell line. The backbone of the gene replacement vector (pCμCδpal) consists of pSV2neo sequences bounded on one side by homology to the μ gene constant (Cμ) region and on the other side by homology to the δ gene constant (Cδ) region. The Cμ and Cδ flanking arms of homology were marked by insertions of an identical 30-bp palindrome which frequently escapes mismatch repair when in heteroduplex DNA (hDNA). As a result, intermediates bearing unrepaired hDNA generate mixed (sectored) recombinants following DNA replication and cell division. To monitor the presence and position of sectored sites and, hence, hDNA formation during the recombination process, the palindrome contained a unique NotI site that replaced an endogenous restriction enzyme site at each marker position in the vector-borne Cμ and Cδ regions. Gene replacement was studied under conditions which permitted the efficient recovery of the product(s) of individual recombination events. Analysis of marker segregation patterns in independent recombinants revealed that extensive hDNA was formed within the Cμ and Cδ regions. In several recombinants, palindrome markers in the Cμ and Cδ regions resided on opposite DNA strands (trans configuration). These results are consistent with the mammalian gene replacement reaction involving two crossing-over events in homologous flanking DNA.

Mechanisms Involved in Targeted Gene Replacement in Mammalian Cells

The “ends-out” or omega (Ω)-form gene replacement vector is used routinely to perform targeted genome modification in a variety of species and has the potential to be an effective vehicle for gene therapy. However, in mammalian cells, the frequency of this reaction is low and the mechanism unknown. Understanding molecular features associated with gene replacement is important and may lead to an increase in the efficiency of the process. In this study, we investigated gene replacement in mammalian cells using a powerful assay system that permits efficient recovery of the product(s) of individual recombination events at the haploid, chromosomal μ-δ locus in a murine hybridoma cell line. The results showed that (i) heteroduplex DNA (hDNA) is formed during mammalian gene replacement; (ii) mismatches in hDNA are usually efficiently repaired before DNA replication and cell division; (iii) the gene replacement reaction occurs with fidelity; (iv) the presence of multiple markers in one homologous flanking arm in the replacement vector did not affect the efficiency of gene replacement; and (v) in comparison to a genomic fragment bearing contiguous homology to the chromosomal target, gene targeting was only slightly inhibited by internal heterology (pSV2neo sequences) in the replacement vector.