Identification of a zebrafish sat1 .a mutant / 中国实验动物学报

Objective In our previous study, we had generated various zebrafish mutant lines with tissue?specific GFP expression by Tol2 transposon?mediated insertional mutagenesis. Among these mutants,the Tol2:20141221t line ex?presses GFP in nervous system, while the position within zebrafish genome where transposon inserted has not yet been iden?tified. The aim of this study was to identify and analyze this genetically modified mutant. Methods The transgenic inser?tion loci in the genome of Tol2:20141221t line was identified byTAIL?PCR and the spatial and temporal expression profile of the affected gene was examined by in situ hybridization. Homozygous mutant of Tol2:20141221t was generated for explo?ring related developmental defects. Results Tol2 transposon was inserted into the 8th intron region of sat1.a gene, and in?duced premature transcription termination. The maternal and zygotic mutants of Tol2:20141221t was generated, while with?out apparent developmental defects. Conclusions We have generated and identified the zebrafishsat1.a mutant mediated by Tol2 transposon. This gene insertion mutant exhibits no obvious developmental abnormalities, but may serve as a power?ful tool to study the development of nervous system.

Identification of zebrafish shield organizer-specific genes / 中国实验动物学报

Objective During zebrafish gastrulation, dramatic movements rearrange cells into three germ layers and contribute to the formation of the shield organizer, which acts as a dorsal signal center to pattern the body axis.Global identification of shield organizer-specific genes in early gastrulas will be valuable for studying the regulatory cascades during organizer formation and body axis establishment.Methods Tg( gsc:GFP) transgenic embryos express GFP in the shield organizer, which is controlled by a 1.8 kb gsc promoter.Flow cytometry technology and RNA deep sequencing analysis were applied to isolate GFP positive cells from the Tg( gsc:GFP) transgenic embryos and systematically uncover the genes highly expressed in the dorsal organizer.Subsequently, the expression of shield organizer-specific genes was further con-firmed by quantitative real-time PCR and whole mount in situ hybridization method during zebrafish embryonic develop-ment.Results GFP-positive cells exceeding 96%purity were isolated from shield-stage Tg(gsc:GFP) transgenic embryos and 657 organizer highly expressed genes were identified through RNA deep sequencing analysis.The results of in situ hy-bridization experiments revealed that a number of genes including KIAA1324, ripply1, twist2, isthmin1, nme4, zgc174153 and rrbp1b were expressed in shield organizer during zebrafish gastrulation.Conclusions The identification of these shield organizer-specific genes in the current study provides useful clues to explore the zebrafish developmental functions in further studies.