Functional expression of the genomic DNA sequences encoding mouse Na,K-ATPase alpha 1 gene by cotransfection of overlapping genomic DNA segments.
The entire 33-kb coding region of the mouse Na,K-ATPase alpha 1 subunit gene was cloned in two overlapping cosmids which contain inserts of 40 kb. To assess the functional expression of the mouse alpha 1 gene, the two cosmids were cotransfected into ouabain-sensitive CV-1 monkey cells yielding an average of 64 resistant colonies per 10(6) cells per microgram of DNA. Analysis of the DNA transferred to the ouabain-resistant transformants by the two cosmids suggests that the generation of a functional gene can occur by homologous recombination between the two introduced segments, as demonstrated by generation of a novel diagnostic restriction fragment. The ability to reconstruct the intact mouse alpha 1 gene in a heterologous host cell and to monitor its functional expression with a selection protocol permits direct identification and isolation of regulatory sequences for the gene.
Tam
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Center for Cancer Research, Massachusetts Institute of Technology, Cambridge 02139.
Geissler
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Graw
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Housman
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D000818:Animals; D015139:Blotting, Southern; D002460:Cell Line; D003001:Cloning, Molecular; D003360:Cosmids; D004247:DNA / Q000235:genetics*; D004351:Drug Resistance; D015870:Gene Expression; D015698:Genomic Library; D051379:Mice; D008815:Mice, Inbred Strains; D009693:Nucleic Acid Hybridization; D010042:Ouabain / Q000494:pharmacology; D015183:Restriction Mapping; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics*; D014162:Transfection
D010042:Ouabain; D004247:DNA; D000254:Sodium-Potassium-Exchanging ATPase