Alternative splicing attenuates transgenic expression directed by the apolipoprotein E promoter-enhancer based expression vector pLIV11.
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.
Cheng
Dongmei
D
Department of Pathology Section on Lipid Sciences, Wake Forest University School of Medicine, Winston Salem, NC 27157, USA.
MacArthur
Philip S
PS
Rong
Shunxing
S
Parks
John S
JS
Shelness
Gregory S
GS
D017398:Alternative Splicing; D000818:Animals; D001057:Apolipoproteins E / Q000235:genetics*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D004330:Drosophila; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D056945:Hep G2 Cells; D006801:Humans; D008099:Liver / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D009928:Organ Specificity; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism; D013045:Species Specificity; D019076:Transgenes
D001057:Apolipoproteins E; D002352:Carrier Proteins; D029721:Drosophila Proteins; D012333:RNA, Messenger; C106092:microsomal triglyceride transfer protein