Ectopic gene targeting exhibits a bimodal distribution of integration in murine cells, indicating that both intra- and interchromosomal sites are accessible to the targeting vector.
Ectopic gene targeting is an alternative outcome of the gene targeting process in which the targeting vector acquires sequences from the genomic target but proceeds to integrate elsewhere in the genome. Using two-color fluorescent in situ hybridization analysis, we have determined the integration sites of the gene targeting vector with respect to the target locus in a murine fibroblast line (LTA). We found that for ectopic gene targeting the distribution of integration sites was bimodal, being either within 3 Mb of the target or on chromosomes distinct from the chromosome carrying the target locus. Inter- and intrachromosomal sites appeared to be equally accessible to the targeting vector, with site-specific variations. Interestingly, interphase analysis indicated that vector sequences which had integrated ectopically in chromosomes other than the target colocalized with the target locus at a significant frequency compared to that of colocalization to random unlinked loci. We propose that ectopic gene targeting could be used to determine which chromosomal domains within the genome are accessible to a given genetic locus. Thus, recombination access mapping may present a new paradigm for the analysis of DNA accessibility and interaction within the genome.
Dellaire
G
G
Institut du cancer de Montréal, Centre de Recherche Louis-Charles Simard, Quebec, Canada.
Lemieux
N
N
Belmaaza
A
A
Chartrand
P
P
D000818:Animals; D002460:Cell Line; D002874:Chromosome Mapping; D002875:Chromosomes / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D005347:Fibroblasts / Q000378:metabolism; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors; D017404:In Situ Hybridization, Fluorescence; D051379:Mice; D008958:Models, Molecular; D015183:Restriction Mapping