NDLI: Alternative splicing attenuates transgenic expression directed by the apolipoprotein E promoter-enhancer based expression vector pLIV11.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Cheng, Dongmei MacArthur, Philip S. Rong, Shunxing Parks, John S. Shelness, Gregory S.
Description	Country affiliation: United States Author Affiliation: Cheng D ( Department of Pathology Section on Lipid Sciences, Wake Forest University School of Medicine, Winston Salem, NC 27157, USA.)
Abstract	The plasmid vector pLIV11 is used commonly to achieve liver-specific expression of genes of interest in transgenic mice and rabbits. Expression is driven by the human apolipoprotein (apo)E 5' proximal promoter, which includes 5 kb of upstream sequence, exon 1, intron 1, and 5 bp of exon 2. A 3.8 kb 3' hepatic control region, derived from a region approximately 18 kb downstream of the apoE gene, enhances liver-specific expression. Here, we report that cDNA sequences inserted into the multiple cloning site (MCS) of pLIV11, which is positioned just downstream of truncated exon 2, can cause exon 2 skipping. Hence, splicing is displaced to downstream cryptic 3' splice acceptor sites causing deletion of cloned 5' untranslated mRNA sequences and, in some cases, deletion of the 5' end of an open reading frame. To prevent use of cryptic splice sites, the pLIV11 vector was modified with an engineered 3' splice acceptor site inserted immediately downstream of truncated apoE exon 2. Presence of this sequence fully shifted splicing of exon 1 from the native intron 1-exon 2 splice acceptor site to the engineered site. This finding confirmed that sequences inserted into the MCS of the vector pLIV11 can affect exon 2 recognition and provides a strategy to protect cloned sequences from alternative splicing and possible attenuation of transgenic expression.
File Format	HTM / HTML
ISSN	00222275
e-ISSN	15397262
DOI	10.1194/jlr.D002709
Journal	The Journal of Lipid Research
Issue Number	4
Volume Number	51
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	2010-04-01
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Discipline Biochemistry Alternative Splicing Apolipoproteins E Genetics Enhancer Elements, Genetic Genetic Engineering Genetic Vectors Promoter Regions, Genetic Transgenes Animals Carrier Proteins Metabolism Drosophila Drosophila Proteins Gene Expression Regulation Hep G2 Cells Liver Mice Mice, Transgenic Organ Specificity Plasmids Chemistry Rna, Messenger Species Specificity Research Support, N.i.h., Extramural
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Endocrinology

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Alternative splicing attenuates transgenic expression directed by the apolipoprotein E promoter-enhancer based expression vector pLIV11[S]

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Alternative splicing attenuates transgenic expression directed by the apolipoprotein E promoter-enhancer based expression vector pLIV11[S]

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