Efficient cDNA cloning by direct phenotypic correction of a mutant human cell line (HPRT-) using an Epstein-Barr virus-derived cDNA expression vector.
Human cells are, in general, poor recipients of foreign DNA, which has severely hampered the cloning of genes by direct phenotypic correction of deficient human cell lines after DNA mediated gene transfer. In this communication a methodology is presented which largely circumvents this problems. The method relies on the use of a recently developed episomal Epstein-Barr-virus-derived cDNA expression vector (Belt et al. (1989) Gene 84, 407-417). The cloning of hypoxanthine phosphoribosyltransferase (HPRT) cDNA, corresponding to a low abundant mRNA in wild type cells is used as a model system. Size fractionated poly (A)+ RNA from wild type cells, which resulted in an approximately 10 fold enrichment in HPRT mRNA, was used to construct a cDNA library of 25,000 independent clones in the pECV25 vector. An HPRT deficient human cell line was transfected and subsequently selected with hygromycin B for DNA uptake. In a small scale experiment only 7000 hygromycin BR transfectants were sufficient to isolate 2 independent HATR clones which were shown to replicate episomes harbouring HPRT cDNA. The first insert had a 5' untranslated region (UTR) and a 3' UTR perfectly in agreement with published data. The second cDNA clone harboured an unusually long 5' UTR and a shorter 3' UTR due to alternative polyadenylation of the HPRT transcript which has not been previously recognized.
Belt
P B
PB
Department of Biochemistry, Gorlaeus Laboratories, Leiden University, The Netherlands.
Jongmans
W
W
de Wit
J
J
Hoeijmakers
J H
JH
van de Putte
P
P
Backendorf
C
C
D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D004351:Drug Resistance / Q000235:genetics; D005822:Genetic Vectors; D004854:Herpesvirus 4, Human / Q000235:genetics*; D006801:Humans; D006921:Hygromycin B; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D008969:Molecular Sequence Data; D010641:Phenotype; D010957:Plasmids; D011061:Poly A / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D011061:Poly A; D006921:Hygromycin B; D004247:DNA; D007041:Hypoxanthine Phosphoribosyltransferase