Recombinant selection by microinjection: a simple cDNA cloning procedure for production of exclusively sense RNA transcripts.
A new strategy for cDNA cloning is presented, designed particularly for identification of recombinants by functional analysis, after microinjection into somatic cells. First-strand synthesis is primed by the oligodeoxyribonucleotide: (formula; see text) After second-strand synthesis and blunting, double-stranded cDNA is formed, which carries restriction sites for NotI and ApaI downstream from the coding sequence. The cDNA is ligated into a plasmid, between two promoters for phage T7 and T3 RNA polymerases. Following transfection and amplification in Escherichia coli, plasmids are extracted from the library or sublibraries. Linearisation with NotI, prior to in vitro transcription, cleaves the plasmids between the 3'-end of the coding sequence and the adjacent promoter and thus ensures that only sense RNAs, suitable for microinjection, are produced after addition of the RNA polymerases. Use of NotI, a rare cutter in the human genome, should ensure that the cDNA inserts are not damaged during linearisation. In the unlikely event that this does happen, a site for ApaI is also available. The method is demonstrated for the human adenine phosphoribosyltransferase-encoding gene.
Digweed
M
M
Institut für Humangenetik, Freie Universität Berlin, F.R.G.
Günthert
U
U
D000228:Adenine Phosphoribosyltransferase / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D004274:DNA, Recombinant; D005347:Fibroblasts; D006801:Humans; D008845:Microinjections; D014158:Transcription, Genetic
D004274:DNA, Recombinant; D000228:Adenine Phosphoribosyltransferase