RRC ID 48837
著者 Shimizu A, Shimizu N.
タイトル Dual promoter expression system with insulator ensures a stringent tissue-specific regulation of two reporter genes in the transgenic fish.
ジャーナル Transgenic Res
Abstract The precise control of spatiotemporal expression of target genes is crucial when establishing transgenic animals, and the introduction of genes for fluorescent marker proteins is inevitable for accelerating research at molecular levels. To assist this, we constructed a novel dual promoter expression vector for two independent reporter genes, green fluorescent protein (GFP) and red fluorescent protein (mCherry). Their expression is designed under the control of two distinct tissue-specific promoters, e.g. zebrafish cardiac muscle-specific promoter (cmlc2) and medaka skeletal muscle-specific promoter (myl2) derived from the myosin light chain 2 genes, and they are placed in a head-to-head orientation. After microinjecting the dual promoter expression vector into fertilized eggs of medaka, the developing fish embryos and the resulting transgenic fish lines showed strong GFP signal in the whole body (skeletal muscle) and mCherry signal in the heart (cardiac muscle). However, weak GFP signal was observed in the heart, indicating a leakiness of the skeletal muscle promoter. To improve the stringency of dual promoter expression, we inserted two chicken-derived insulators, e.g. tandem copies of the core sequence (250 bp) of cHS4 (5'-hypersensitive site-4 chicken beta-globin insulator), in the boundary of two promoters. The dual promoter expression vector with insulator now ensured the stringent tissue-specific expression in the transgenic fish lines. Thus, our dual promoter expression system with insulator is compatible to the conventional IRES and fused reporter gene systems and will be an alternative method to produce the transgenic fishes.
巻・号 22(2)
ページ 435-44
公開日 2013-4-1
DOI 10.1007/s11248-012-9653-8
PMID 22983842
MeSH Animals Animals, Genetically Modified / growth & development* Chickens / genetics Gene Expression Regulation, Developmental Green Fluorescent Proteins / genetics* Insulator Elements / genetics Luminescent Proteins / genetics* Organ Specificity / genetics* Oryzias / genetics Promoter Regions, Genetic* Zebrafish / genetics Zebrafish / growth & development
IF 1.856
引用数 9
WOS 分野 BIOTECHNOLOGY & APPLIED MICROBIOLOGY BIOCHEMICAL RESEARCH METHODS BIOCHEMISTRY & MOLECULAR BIOLOGY
リソース情報
メダカ pIST4-cmlc2::mCherry-HBA0::GFP (TG935) pIST4-cmlc2::mCherry-myl2::GFP (TG941) pIST6-cmlc2::mCherry-myl2::GFP (TG955)