PiggyBac Transposon-mediated Gene Trap Approach Identifies Regulated Genes In Zebrafish

With the completion of zebrafish genome sequencing, it was found that the homology ratio of zebrafish genes and human genes was up to87%, making zebrafish an ideal animal model for the study of human genes.Transposons have been widelyly used for genetic manipulations in lower organisms and played important roles in the study of gene functions in these animals. Nowdays Tol2transposon system, combined with gene trap method are mainly used in zebrafish mutant screening. Tol2integrates as a single copy through a cut-and-paste mechanism, and it does not cause any rearrangement or modification at the target site except for the creation of8base pair duplication. Therefore,8bp direct repeats are always seen adjacent to integrated Tol2elements. Tol2often leaves a small deletion or insertion after excision from the original site, making it difficult to do reverse mutations.PiggyBac (PB), a DNA transposon from the cabbage looper moth Trichoplusia ni, is a mobile genetic element that efficiently transposes between vectors and chromosomes via a "cut and paste" mechanism. During transposition, the PB transposase recognizes transposon-specific inverted terminal repeat sequences (ITRs) located on both ends of the transposon vector and efficiently moves the contents from the original sites and efficiently integrates them into TTAA chromosomal sites. PB elements have been used to transform the germline of more than a dozen species spanning four orders of insects. In2005, Ding tested the ability of piggyBac (PB) to transpose in mammalian systems. He showed that PB elements carrying multiple genes could efficiently transpose in human and mouse cell lines and also in mice. In2006, Fraser demonstrated that PB transposon was capable of transposition in primate cells and embryos of zebrafish. These results validate piggyBac as a valuable tool for genetic analysis in zebrafish.PiggyBac transposition System, including helper plasmid-pCMV-hyPBase and donor plasmid-5’-PTK-3’was recombined to form a binary co-transfection assay system.Construction of heper plasmid pCMV-hyPBase-IRES-EGFP was as follows: the IRES-EGFP fragment from pPRIG plasmid was cloned into the XbaI and XhoI sites of pCMV-hyPBase. Construction of heper plasmid pZPC0.5-hyPBase-IRES-EGFP was as follows:ZPC0.5promoter was PCR amplified from zebrafish genomic DNA and cloned into the SpeI and EcoRI sites of pCMV-hyPBase-IRES-EGFP to replace CMV promoter.Construction of donor plasmid PTK-mCherry (gene trap vector)was as follows:the mCherry fragment from pmCherry-N1plasmid was cloned into the Bell and NcoI sites of5’-PTK-3’ Construction of plasmid pCS2+-hyPBase (used as DNA template for in vitro synthesized transposase mRNA) was as follows:the hyPBase fragment from pCMV-hyPBase plasmid was cloned into the EcoRl and XhoI sites of pCS2+.Linearized pCMV-hyPBase-IRES-EGFP plasmid was microinjected into the cytoplasm of one-cell stage zebrafish embryos.Transgenic zebrafish were obtained by detecting the ubiquitous expression of EGFP in FO zebrafish larvae.The plasmid inserted in the genome could be transmitted to the F1progeny.Regulated gene expression by CMV promoter could be recapitulated in transgenic fish and pCMV-hyPBase-IRES-EGFP transgenic zebrafish (founder) was obtained.In vitro synthesized transposase hyPBase mRNA and donor plasmid PTK-mCherry were coinjected into one-cell stage embryos and obtained fish expressing mCherry in different temporally and spatially patterns.The8%mCherry expression rate in FO zebrafish proved the effectiveness of piggyBac transposon as gene trap tools in zebrafish.Helper transgenic zebrafish containing the transposase gene and donor transgenic zebrafish containing many single-copy PB insertions will be out-crossed and in vivo transposition system using PB will be developed using Double-transgenic fish containing the transposase gene and single-copy PB insertions.We propose our gene trap approach should facilitate studies of vertebrate development and organogenesis. Thymus is considered as a key organ of the immune system in vertebrates. Several key transcription factors such as pax1, pax9, eya1、six1、six4, foxnl have been identified that are critical for various aspects of thymus organogenesis. Amphioxus or lancele, a cephalochordate, located at the base of vertebrates, and is becoming an emerging model organism for insights into the origin and evolution of vertebrates. It has been considered that Amphioxus, as cephalochordate, lacks adaptive immune system. However, recent researches on the amphioxus has shown that some homologous or similar genes of many pivotal genes in vertebrate adaptive immune system, such as pax1, pax9, eya1、six1、six4, foxn1, also existed in Amphioxus.Using bioinformatics analysis, we found that several genes such as paxl, pax9, eya1、six1、six4, foxn1, which were critical for thymus organogenesis in vertebrates, also had homologoues in the Florida lancelet database.In case of high similarities between Branchiostoma floridae and Branchiostoma belcheri genes, primers were designed using Branchiostoma floridae gene sequences as templates. RT-PCR was used and we get Branchiostoma belcheri six1gene fragment (with complete CDS) and six4gene fragment; The predicted protein of Branchiostoma belcheri Sixl is200amino acids in length,with theoretical molecular weight of22057.6Da and isoclcctric point of7.84.It is a hydrophilic protein that belongs to the homeodomain superfamily.