Construction of multi-purpose vectors, pCNS and pCNS-D2, are suitable for collection and functional study of large-scale cDNAs.
To efficiently perform collection and functional studies of large-scale cDNAs, we constructed multi-functional cDNA vectors for efficient full-length cDNA cloning, direct sequencing, easy screening, and the expression of cDNA in vitro and in vivo without subcloning the cDNA into other vectors. The constructed vectors, pCNS and pCNS-D2, contain a multi-cloning site for uni-directional full-length cDNA cloning, T7 and Sp6 RNA polymerase promoters for in vitro transcription and translation, and hCMV immediate early promoter and BGH poly(A) to allow expression in mammalian cells. Using these vectors, we constructed full-length enriched cDNA libraries containing 60-75% of the full-length cDNAs using two different oligo-capping methods. The subtracted cDNA libraries could also be constructed by removing of EF1-alpha cDNA, a highly expressed cDNA. In addition, we confirmed the translation of EF1-alpha cDNA in vitro and the expression of luciferase cDNA in mammalian cells. The expression efficiency of luciferase cDNA in different cell lines, such as HeLa, Hep3B, SNU638, and SNU668, showed that pCNS vectors can highly express target genes in different cell types. These results indicated that our multi-purpose vectors, pCNS and pCNS-D2, are useful tools for the construction of full-length cDNA libraries and high-throughput based functional study of cDNAs.
Oh
Jung-Hwa
JH
Laboratory of Human Genomics, Division of Genomics and Proteomics, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-333, Republic of Korea.
Sohn
Ho-Yong
HY
Kim
Jeong-Min
JM
Kim
Yong Sung
YS
Kim
Nam-Soon
NS
D000818:Animals; D001483:Base Sequence; D045744:Cell Line, Tumor; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D015723:Gene Library; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D020648:Peptide Elongation Factor 1 / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D018076:DNA, Complementary; D020648:Peptide Elongation Factor 1; D008156:Luciferases