The molecular basis of multiple vector insertion by gene targeting in mammalian cells

Gene targeting using sequence insertion vectors generally results in integr ation of one copy of the targeting vector generating a tandem duplication o f the cognate chromosomal region of homology. However, occasionally the tar get locus is found to contain > 1 copy of the integrated vector. The mechan ism by which the latter recombinants arise is not known. In the present stu dy, we investigated the molecular basis by which multiple vectors become in tegrated at the chromosomal immunoglobulin mu locus in a murine hybridoma. To accomplish this, specially designed insertion vectors were constructed t hat included sis diagnostic restriction enzyme markers in the C mu region o f homology to the target chromosomal mu locus. This enabled contributions b y the vector-borne and chromosomal C mu, sequences at the recombinant locus to be ascertained. Targeted recombinants were isolated and analyzed to det ermine the number of vector copies integrated at the chromosomal immunoglob ulin mu locus. Targeted recombinants identified as bearing > 1 copy of the integrated vector resulted from a C mu triplication formed by two vector co pies in tandem. Examination of the fate of the C mu region markers suggeste d that this class of recombinant was generated predominantly, if not exclus ively, by two targeted vector integration events, each involving insertion of a single copy of the vector. Both vector insertion events into the chrom osomal mu locus were consistent with the double-strand-break repair mechani sm of homologous recombination. We interpret our results, taken together, t o mean that a proportion of recipient cells is in a predetermined state tha t is amenable to targeted but not random vector integration.