Generation and phenotype analysis of heat-induced Vg1 transgene amphioxus strain
[Objective]Vg1,an important ligand of the Nodal signaling pathway,is involved in dorsal-ventral and left-right axis patterning during the early embryogenesis of vertebrates.Cephalochordate amphioxus,the most basic divergent group of the Chordata,exhibits a body pattern similar but simpler than that of vertebrates.The amphioxus genome contains only one Vg1 gene.Similar to vertebrates,the amphioxus Vg1 gene is important in the establishment of the dorso-ventral and left-right body axes during amphioxus embryonic development.Current methods used to study the function of the amphioxus Vg1 gene are limited to in situ hybridization,gene knockout,and injection of mRNA into unfertilized eggs,but all with their own limitations.Genes often perform different functions when expressed at different stages of embryonic development.Therefore,it is imperative to establish a method that can control the expression of the transferred gene at the required moment to reveal the gene function at different stages.[Methods]Amphioxus(Branchiostoma floridae)were initially acquired from Jr-Kai Yu,at the Institute of Cellular and Organismic Biology,Academia Sinica,Taipei,Taiwan,China,and the colony was maintained under previously described conditions.Gametes were induced using thermal induction combined with photoperiod control.The vector backbone was linearized using double digestion and subsequently recovered.The molecular cloning method was used to obtain the Hsp70 promoter and the coding sequence of Vg1 gene.The transgenic plasmid was constructed using homologous recombination ligation.Tol2 transposase mRNA was synthesized in vitro using the mMESSAGE mMACHINETM T3 Transcription Kit.The injection solution was prepared containing 20％(volume fraction)glycerol,50 mg/mL Oregon Green dextrans,and a total of 70％(volume fraction)of the transgenic plasmid plus Tol2 mRNA,which was injected into amphioxus unfertilized eggs and selected to obtain transgenic F0.Transgenic F0 was intensively cultured to sexual maturity and induced to produce gametes.After fertilization,embryos were cultured at 25 ℃ until them reached a specific stage,and some embryos were taken for heat shock.After heat shock,they were returned to 25 ℃ for continued culturing.Embryos at the expected developmental stages were fixed in 4％(mass fraction)perfluoroalkoxy alkane-3-(morpholino)propanesulfonic acid(PFA-MOPS)and then stored in 70％(volume fraction)ethanol at-20 ℃ until needed.Whole-mount in situ hybridization was used for gene expression analysis in transgenic F1 embryos.An inverted Olympus microscope was used for embryo observation and photography.PCR identification was used to illustrate the correlation between specific phenotypes and PCR positivity.[Results]Heat shock treatment of Vg1 transgenic amphioxus F1 embryos at the late blastula and mid-gastrula stages respectively resulted in an abnormal formation of the dorsoventral axis and symmetrical distribution of left and right organs.A certain number of individuals with abnormal phenotypes and individuals with normal phenotypes were selected for PCR identification.The results showed that individuals with abnormal phenotypes were all PCR positive,while individuals with normal phenotypes were all PCR negative.Correlated with the above phenotypes,gene expression analysis results showed that in some F1 embryos heat-shocked at late blastula stage,Vg1 was expressed systemically.The expression of the dorsal mesoderm marker gene Gsc was expanded,the expression of the abdominal marker gene Evx was down-regulated,and the neural marker gene SoxB1a spread ventrally.In some F1 embryos heat-shocked at mid-gastrula stage,the left-specific gene Pitx and the left preoral pit marker gene Pit were expressed symmetrically on both side.[Conclusion]We have generated a stable transgenic amphioxus strain carrying heat-induced Vg1 expression through a thermosensitive Hsp70 promoter during embryonic development.This transgenic strain circumvents some of the shortcomings associated with current amphioxus gene function research methods.At the same time,it provides a new method for studying the function of Vg1 at different embryonic development stages of amphioxus and also serves as a reference for the subsequent construction of other transgenic strains with timely activation systems.
国家科技期刊平台
NSTL
万方数据
维普
