The Identification Of The Zebrafish Mutation Of The Heart Developmental Candidate Genes POP3 And The Related Functional Studies

Zebrafish has become one of the most important vertebrate models in developmental biology, but the gene knockout method in zebrafish has not yet been well established, which prevents the fully elucidation of biological functions of zebrafish genes.To our gratified, a novel gene knock-out technology was developed in 2008, the OPEN (Oligomerized pool engineering) technology, which is a method of designing high affinity zinc-finger nuclease for gene target. This technology can knockout genes in vivo quickly and efficiently and creat knockout strains. The laboratory has screened out the 355 bp target sites of zebrafish POP3 gene and has constructed two corresponding plasmids of the zinc finger protein half site. In this project, the plasmid has been transcribed into RNA, then the RNA was injected into the 1 cells of zebrafish embryo. When the embryos developed to 24h, we collected them and extracted the whole genome, which was used to be PCR template to amplify the target gene with either side of PCR primer. After preliminary identification of PCR products with some special enzyme, the product was connected with pMD18-T to be sequenced in order to detect the effectiveness of OPEN knock-out technology.Recently, there is a new method to knockdown a target geen-TALEN(transcription activator-like (TAL) effector nucleases) targeted gene knock out technology, It has many advantages like that people can construct module according to whatever target genes they want to choose, in other word, that is, TALE is more flexible in module construction of target sequence.This article also used the new molecular tools. Though on-line analysis of TALEN target sites of POP3 gene, this article used the Xanthomonas’ TAL single module to obtain the most appropriate site and constructed the recombine nuclease plasmid which aimed directly to the first exon of POP3 nucleic acid sequences in order to interrupt and knockout the taget gene. Then we detected the recombinate TALEN plasmids’effectiveness of knockout.In addition, our laboratory has some other research results showing that POP3 and ATF4 have interaction in vitro.In order to study the interaction of POP3 and ATF4 in zebrafish, we marked the POP3 and ATF4 primers with flag or myc label respectively by PCR, then recycled the PCR products as template to obtain mRNA which has flag or myc tag. After that, we injected them into 4 to 8-cell of zebrafish embryo commonly. When they developed to Bud, collected the embryos and extracted their proteins to do the Co-Immunoprecipitation experiment. The result of it pointed out that POP3 and ATF4 do have interactions with each other in zebrafish.