Construction Of Heart Developmental Candidate Genes CFL And CXXC5TALEN-Knockout Mutants

Zebrafish, also known as the blue strip fish, strip fish and brachydanio rerio, is native to India, Bangladesh. Zebrafish has many advantages like individual small, breeding cost less and strong regenerative ability,so zebrafish has been widely used as one of the most important vertebrate model organism in developmental biology. However, due to the zebrafish embryonic stem cell technology is not mature enough, it does not achieve gene targeting by ES ways like the mouse. It has always been sparing no effort to try to be prepared by a variety of ways/screening mutant in zebrafish in the last ten years, and has gradually established a variety of methods, including ENU (N-ethyl-N-nitrosourea) chemical mutagenesis, reverse transcription virus insertion mutagenesis, gene trapping and ZFN (Zinc finger nuclease)-mediated site-directed mutations. However, these methods have their limitations, they did not achieve any of zebrafish genes were targeted modification.Design and construction of TALEN(transcription activator-like (TAL) effector nucleases),which rised in recent three or four years, is based on a transcription activator-like effector secreted by Xanthomonas that is plant pathogen.Fusing the DNA-binding domain and the FokI endonuclease enzyme cutting domain of TALE proteins, it can induce double-strand breaks at the specific site and trigger the cell’s own DNA repair mechanisms (Non-homologous end joining and Homologus Recombination),then,it will introduce the gene mutation,gene modification and knock in, thus realizing targeted genome editing.accordingly,Achieve genome directional edit TALENs success in many model organisms.In addition,TALEN 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. Our laboratory has discovered two genes associated with heart development by previous work, namely CFL and CXXC5. They both contain a CXXC domain, belonging high degree of homology to two genes in zebrafish, and in higher vertebrates exist only CXXC5genes. Preliminary results confirmed the CFL and early CXXC5gene plays an important role in early heart development.In order to make the results more convincing, this paper intends to use TALEN targeting technical knockout CFL and CXXC5in zebrafish and tries to build a CFL and CXXC5TALEN targeting gene knockout strains and then study its related effects. Details are as follows: To begin with,the suitable target sites of CFL and CXXC5were chosen through the corresponding website.And then based on pCS2-PEAS and pCS2-PERR expression vector, we constructed the left and right half sites recombinant nuclease plasmids of CFL and CXXC5genes.Finally,the constructed expression plasmids were transcribed into mRNA which was then microinjected into zebrafish1cell phase embryos. After24hpf-48hpf incubation, we extracted the genome of embryos and used PCR amplification and then enzyme digestion to test the effectiveness of the recombinant expression plasmid. At present, a preliminary screening of the CFL and CXXC5genes has been completed and the the further work is under way.