Physical mapping of genes in somatic cell radiation hybrids by comparative genomic hybridization to cDNA microarrays.
BACKGROUND
Somatic cell mutants can be informative in the analysis of a wide variety of cellular processes. The use of map-based positional cloning strategies in somatic cell hybrids to analyze genes responsible for recessive mutant phenotypes is often tedious, however, and remains a major obstacle in somatic cell genetics. To fulfill the need for more efficient gene mapping in somatic cell mutants, we have developed a new DNA microarray comparative genomic hybridization (array-CGH) method that can rapidly and efficiently map the physical location of genes complementing somatic cell mutants to a small candidate genomic region. Here we report experiments that establish the validity and efficacy of the methodology.


RESULTS
CHO cells deficient for hypoxanthine:guanine phosphoribosyl transferase (HPRT) were fused with irradiated normal human fibroblasts and subjected to HAT selection. Cy5-labeled genomic DNA from the surviving hybrids containing the HPRT gene was mixed with Cy3-labeled genomic DNA from normal CHO cells and hybridized to a microarray containing 40,185 cDNAs, representing 29,399 genes (UniGene clusters). The DNA spots with the highest Cy5:Cy3 fluorescence ratios corresponded to a group of genes mapping within a 1 Mb interval centered near position 142.7 Mb on the X chromosome, the genomic location of HPRT.


CONCLUSIONS
The results indicate that our physical mapping method based on radiation hybrids and array-CGH should significantly enhance the speed and efficiency of positional cloning in somatic cell genetics.
Lin
Johann Y
JY
Department of Vascular Biology, The Scripps Research Institute, 10550 N, Torrey Pines Road, La Jolla, CA 92037, USA. Ginsberg@scripps.edu
Pollack
Jonathan R
JR
Chou
Fan-Li
FL
Rees
Christian A
CA
Christian
Allen T
AT
Bedford
Joel S
JS
Brown
Patrick O
PO
Ginsberg
Mark H
MH
D000818:Animals; D016466:CHO Cells / Q000737:chemistry / Q000378:metabolism; D002460:Cell Line; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics*; D005347:Fibroblasts / Q000737:chemistry / Q000378:metabolism; D005796:Genes / Q000235:genetics*; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000172:deficiency / Q000235:genetics; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D020161:Physical Chromosome Mapping / Q000379:methods*; D021101:Radiation Hybrid Mapping / Q000379:methods*
D018076:DNA, Complementary; D007041:Hypoxanthine Phosphoribosyltransferase