Plasmids for the cloning and expression of full-length double-stranded cDNAs under control of the SV40 early or late gene promoter.
Okayama and Berg (1) have recently described a technique for the high efficiency cloning of full-length dscDNAs. We have constructed eukaryotic expression vectors compatible both with this technique (and with classical techniques) for dscDNA cloning. The vectors are such that recombinants obtained contain dscDNAs in the correct orientation downstream from a block of sequence comprising either the SV40 early or late gene promoter linked to a pair of splice sites from a rabbit beta-globin gene. A sequence encoding an SV40 polyadenylation site follows the dscDNA. We have used our vectors to make a library from chicken oviduct polyA(+) RNA using the Okayama and Berg technique. Ovalbumin recombinants occur in the library at the expected frequency and a high proportion contain full length copies of the ovalbumin mRNA. However, a similar result was not obtained for conalbumin recombinants. When recombinants are introduced into eukaryotic cells by either calcium phosphate coprecipitation or protoplast fusion, expression of chicken ovalbumin or conalbumin may be detected by indirect immunofluorescence. Under optimal conditions (use of SV40 late promoter and cos 7 cells) ovalbumin protein could be detected when the ovalbumin recombinant was present in only 2% of the protoplasts used for fusion. This suggests that colony banks obtained using our vectors could be screened in batches of 50 by protoplast fusion followed by a search for expression of a given protein using indirect immunofluorescence.
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004262:DNA Restriction Enzymes; D005796:Genes; D005814:Genes, Viral; D005822:Genetic Vectors; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D009876:Operon; D010047:Ovalbumin / Q000235:genetics; D010957:Plasmids; D013539:Simian virus 40 / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection
D010047:Ovalbumin; D004247:DNA; D004262:DNA Restriction Enzymes