Tol2 Transposon Mediated-zebrafish Gene Mutagenesis、generation And Identification Of Zebrafish Shield Organizer-specific Genes

Zebrafish as an excellent model organism, the process of embryonic development is participated in and regulated by various genes and signaling pathways. It is very suitable for developmental biology research and genetic disease model building, etc. The mutant as a kind of gene loss of function, is the important tool to study gene function and reveal the mechanism of development. For further to explore the development of zebrafish embryos, reveal the development mechanism and find more research materials for zebrafish embryo development, in this paper, we used the Tol2 transposon-mediated gene trap method to find the lines which GFP (green fluorescent protein) express specifically. And further, have generated the mutants that related to the zebrafish embryo development and Identified which genes was mutated. Moreover, we had a preliminary analysis for this genes play what roles and functions in the process of embryonic development.In this study, we co-inject Tol2 plasmid and mRNA into zebrafish single cell and we have trapped 6 lines express GFP specifically, we generated two of them which express GFP earlier to maternal and zygotic mutant and find that one of them has epibody defective in maternal and homozygous mutant, the other one exhibits no obvious developmental abnormalities, but it express GFP in nervous system specifically may function as a powerful tool to study the development of nervous system.And we find that one of the F2 females generation could generate defective offsprings.Fluorescence distribution of Tol2:20141017m line has some specificity of time and space, this line express fluorescence both in maternal and zygotic stage, expressed in margin area during the shield stage, expressed mainly in the somite and notochord during the segment stage, then expressed in myotome and YSL post fertilization 24 hours. TAIL-PCR analysis showed that the gene trapped by Tol2 transposon in this transgenic family was si:ch73-52/24.4 gene. Data on Ensembl shows that si:ch73-52f24.4 gene have three transcripts, but none of them could encode protein. Tol2 transposon insert into the first intron of two si:ch7S-52f24.4 transcripts blocking the normal expression of these two transcripts, because with the tail structure in transposons. However, the third transcript is not affected from the point of view of location. The maternal and zygotic mutant embryos has obvious flaws in the process of epiboly movement:the time of development was extended, Yolk cannot be fully absorbed, tail bud became flat, part of embryos can’t development into the segment period even, the embryos can development to 24 h normally is shorter of the wild type. So our preliminary analysis is that si:ch73-52f24.4 may be an important maternal lncRNA influencing the process of epiboly movement, it may have very important influence in the differentiation of the layer and segment development.Tol2:20141221t line express fluorescent specifically in the nervous system, mainly concentrate in the brain and upper of spinal chord, we identificated the trapped gene is sat1.a by the 5’RACE. Sat1.a is a kind of the spermine/spermine Nl-acetyl transferase gene, which are three kinds of homologous genes in the zebrafish. Tol2 transposon was inserted into the eighth intron, and induced premature transcription termination. We had not yet observed a consistent developmental defective with the gene, but at the same time we also found that one of four females in the F2 generation can generate offsprings with all spine bent, no matter cross with wild type or homozygote. This mutation seems has nothing to do with the location of fluorescence, we have not made clear whether associated with the gene we identified, if not, which gene was mutated.To sum up, Tol2 transposon mediated gene capture technology is a feasibility of forward genetics research tool. We had successfully captured a lncRNA, which had not reported yet, it provides a new material for our research on lncRNA. Besides, the line expressed in nervous system can serve as a powerful tool for nervous system, provide the reference for researching zebrafish nervous system development.In addition, the author is also involved in identification of zebrafish shield organizer-specific genes: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 Tg(gsc:GFP) transgenic embryos and systematically uncover the genes highly expressed in the dorsal organizer. 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 hybridization experiments revealed a number of genes including isthminl, KIAA1324, rrbp1b, ripply1, twist2, nme4 and zgc174153 are expressed in shield organizer during zebrafish gastrulation. The identification of these shield organizer-specific genes in the current study provides useful clues to explore their developmental functions in next studies.