Construction of multi-purpose vectors, pCNS and pCNS-D2, are suitable for collection and functional study of large-scale cDNAs

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Title
Construction of multi-purpose vectors, pCNS and pCNS-D2, are suitable for collection and functional study of large-scale cDNAs
Author(s)
Jung Hwa Oh; H Y Sohn; Jeong-Min Kim; Yong Sung KimNam-Soon Kim
Bibliographic Citation
Plasmid, vol. 51, no. 3, pp. 217-226
Publication Year
2004
Abstract
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-α cDNA, a highly expressed cDNA. In addition, we confirmed the translation of EF1-α 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.
Keyword
Full-length cDNAGene expression in vivo and in vitroMulti-functional vectorpCNSpCNS-D2Subtraction
ISSN
0147-619X
Publisher
Elsevier
DOI
http://dx.doi.org/10.1016/j.plasmid.2004.01.001
Type
Article
Appears in Collections:
Division of Biomedical Research > Genome Editing Research Center > 1. Journal Articles
Division of Biomedical Research > Rare Disease Research Center > 1. Journal Articles
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