Upstream regulatory region of zebrafish lunatic fringe: isolation and promoter analysis.
Lunatic fringe (Lfng), one modulator of Notch signaling, plays an essential part in demarcation of tissues boundaries during animal early development, especially somitogenesis. To characterize the promoter of zebrafish lfng and generate somite-specific transgenic zebrafish, we isolated the upstream regulatory region of zebrafish lfng by blast search at the Ensembl genome database ( http://www.ensembl.org ) and analyzed the promoter activity using green fluorescent protein (GFP) as a reporter. Promoter activity assay in zebrafish shows that the 0.2-kb fragment containing GC-box, CAAT-box, and TATA-box can direct tissue-specific GFP expression, while the 0.4-kb and 1.2-kb fragments with further upstream sequence included drive GFP expression more efficiently. We produced lfngEGFP-transgenic founders showing somite-specific expression of GFP and consequently generated a hemizygous lfngEGFP-transgenic line. The eggs from lfngEGFP-transgenic female zebrafish show strong GFP expression, which is consistent to the reverse-transcription polymerase chain reaction PCR (RT-PCR) detection of lfng transcripts in the fertilized eggs. This reveals that zebrafish lfng is a maternal factor existing in matured eggs, suggesting that fish somitogenesis may be influenced by maternal factors.
Liu
Jing
J
State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, China.
Sun
Yong-Hua
YH
Wang
Na
N
Wang
Ya-Ping
YP
Zhu
Zuo-Yan
ZY
D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000737:chemistry; D005260:Female; D018507:Gene Expression Regulation, Developmental / Q000502:physiology; D018014:Gene Transfer Techniques; D016695:Glycosyltransferases / Q000235:genetics* / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000096:biosynthesis; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D015027:Zebrafish / Q000235:genetics*; D029961:Zebrafish Proteins / Q000235:genetics* / Q000502:physiology
D017931:DNA Primers; D018076:DNA, Complementary; D029961:Zebrafish Proteins; D049452:Green Fluorescent Proteins; D016695:Glycosyltransferases; C498390:lfng protein, zebrafish