Cloning Expressed cDNAS from Defined Chromosomal Regions Using Interspecific Microcell Hybrids and Subtractive Hybridization
Genes expressed from a defined genomic interval can be cloned using interspecific microcell hybrids and subtractive hybridization. This approach involves identifying a pair of interspecific hybrids that contain nearly isogenic fragments of a relevant chromosome, except for a region of nonoverlap encompassing the locus of interest. Radiolabeled cDNAs are synthesized from the hybrid cell line that contains the region of interest and hybridized in solution with an excess of biotinylated mRNA from the microcell hybrid lacking the region of interest. Subtracted cDNA probes enriched for sequences expressed from the nonoverlap region are isolated by the addition of streptavidin followed by organic extraction. These subtracted cDNA probes are used to screen a cDNA library prepared from an appropriate tissue, and candidate cDNAs are mapped by Southern hybridization to determine whether they are encoded within the nonoverlap region. This technique is an attractive alternative to other methods for identifying genes in a genomic region because it does not require that the gene of interest be localized to a minimal genetic interval, and the genomic DNA encompassing the region need not be cloned. Additionally, the method detects only expressed genes, simplifying the search for candidate genes within a genomic region.
Cerosaletti
KM
Division of Molecular Medicine, Fred Hutchinson Cancer Research Center, 1124 Columbia Street, Seattle, Washington, 98104
Fournier
REK