Document Detail


Effective expression-independent gene trapping and mutagenesis mediated by sleeping beauty transposon.
MedLine Citation:
PMID:  23021550     Owner:  NLM     Status:  In-Data-Review    
Abstract/OtherAbstract:
Expression-independent gene or polyadenylation [poly(A)] trapping is a powerful tool for genome-wide mutagenesis regardless of whether a targeted gene is expressed. Although a number of poly(A)-trap vectors have been developed for the capture and mutation of genes across a vertebrate genome, further efforts are needed to avoid the 3'-terminal insertion bias and the splice donor (SD) read-through, and to improve the mutagenicity. Here, we present a Sleeping Beauty (SB) transposon-based vector that can overcome these limitations through the inclusion of three functional cassettes required for gene-finding, gene-breaking and large-scale mutagenesis, respectively. The functional cassette contained a reporter/selective marker gene driven by a constitutive promoter in front of a strong SD signal and an AU-rich RNA-destabilizing element (ARE), which greatly reduced the SD read-through events, except that the internal ribosomal entry site (IRES) element was introduced in front of the SD signal to overcome the phenomenon of 3'-bias gene trapping. The breaking cassette consisting of an enhanced splicing acceptor (SA), a poly(A) signal coupled with a transcriptional terminator (TT) effectively disrupted the transcription of trapped genes. Moreover, the Hsp70 promoter from tilapia genome was employed to drive the inducible expression of SB11, which allows the conditional remobilization of a trap insert from a non-coding region. The combination of three cassettes led to effective capture and disruption of endogenous genes in HeLa cells. In addition, the Cre/LoxP system was introduced to delete the Hsp70-SB11 cassette for stabilization of trapped gene interruption and biosafety. Thus, this poly(A)-trap vector is an alternative and effective tool for identification and mutation of endogenous genes in cells and animals.
Authors:
Guili Song; Qing Li; Yong Long; Perry B Hackett; Zongbin Cui
Publication Detail:
Type:  Journal Article     Date:  2012-08-23
Journal Detail:
Title:  Journal of genetics and genomics = Yi chuan xue bao     Volume:  39     ISSN:  1673-8527     ISO Abbreviation:  J Genet Genomics     Publication Date:  2012 Sep 
Date Detail:
Created Date:  2012-10-01     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  101304616     Medline TA:  J Genet Genomics     Country:  China    
Other Details:
Languages:  eng     Pagination:  503-20     Citation Subset:  IM    
Copyright Information:
Copyright © 2012. Published by Elsevier Ltd.
Affiliation:
The Key Laboratory of Aquatic Biodiversity and Conservation, Institute of Hydrobiology, Chinese Academy of Sciences, 7 Donghu Rd., Wuhan, Hubei 430072, China; Graduate University of the Chinese Academy of Sciences, Beijing 100049, China.
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