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题名: Effective Expression-Independent Gene Trapping and Mutagenesis Mediated by Sleeping Beauty Transposon
作者: Song, Guili1, 2; Li, Qing1; Long, Yong1; Hackett, Perry B.3; Cui, Zongbin1
通讯作者: Cui, ZB (reprint author), Chinese Acad Sci, Inst Hydrobiol, Key Lab Aquat Biodivers & Conservat, 7 Donghu Rd, Wuhan 430072, Hubei, Peoples R China.
关键词: Poly(A) trapping ; Sleeping Beauty transposon ; Insertional mutagenesis ; HeLa cells ; Zebrafish embryos
刊名: JOURNAL OF GENETICS AND GENOMICS
发表日期: 2012-09-01
DOI: 10.1016/j.jgg.2012.05.010
卷: 39, 期:9, 页:503-520
收录类别: SCI
文章类型: Article
部门归属: [Song, Guili; Li, Qing; Long, Yong; Cui, Zongbin] Chinese Acad Sci, Inst Hydrobiol, Key Lab Aquat Biodivers & Conservat, Wuhan 430072, Hubei, Peoples R China; [Song, Guili] Chinese Acad Sci, Grad Univ, Beijing 100049, Peoples R China; [Hackett, Perry B.] Univ Minnesota, Minneapolis, MN 55455 USA
WOS标题词: Science & Technology ; Life Sciences & Biomedicine
资助者: National Natural Science Foundation of China [30871442]; National Basic Research Program of China [2012CB944500]
类目[WOS]: Biochemistry & Molecular Biology ; Genetics & Heredity
研究领域[WOS]: Biochemistry & Molecular Biology ; Genetics & Heredity
摘要: 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.
英文摘要: 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.
关键词[WOS]: EMBRYONIC STEM-CELLS ; DEVELOPMENTALLY-REGULATED GENES ; MESSENGER-RNA DECAY ; INSERTIONAL MUTAGENESIS ; FUNCTIONAL-ANALYSIS ; LARGE-SCALE ; HEPATOCELLULAR-CARCINOMA ; PIGGYBAC TRANSPOSONS ; MOUSE GENOME ; ELEMENT
语种: 英语
WOS记录号: WOS:000309605900010
ISSN号: 1673-8527
Citation statistics:
内容类型: 期刊论文
URI标识: http://ir.ihb.ac.cn/handle/342005/17266
Appears in Collections:水生生物分子与细胞生物学研究中心_期刊论文

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作者单位: 1.Chinese Acad Sci, Inst Hydrobiol, Key Lab Aquat Biodivers & Conservat, Wuhan 430072, Hubei, Peoples R China
2.Chinese Acad Sci, Grad Univ, Beijing 100049, Peoples R China
3.Univ Minnesota, Minneapolis, MN 55455 USA

Recommended Citation:
Song, Guili; Li, Qing; Long, Yong; Hackett, Perry B.; Cui, Zongbin.Effective Expression-Independent Gene Trapping and Mutagenesis Mediated by Sleeping Beauty Transposon,JOURNAL OF GENETICS AND GENOMICS,2012,39(9):503-520
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