Introduction Of Tol2-mediated Gene Transfer Method Into Amphioxus And Generation Of Two Transgenic Lines

Amphioxus or lancelets are regarded as a promising model for studying developmental and evolutionary mechanisms of vertebrates due to its unique phylogenetic position and simplicity of both anatomy and genome.Although several recent achievements make amphioxus more applicable as a laboratorial model animal,a transgenic method remains to be developed for amphioxus studies.Tol2 transposon system is a useful tool for generating transgenic animals,which was revealed to efficiently carry donor DNA up to 10 kb without impacting its integration efficiency in many vertebrate models.To introduce a Tol2 system-mediated gene transfer method into amphioxus,this report assembled two donor vector pmini-mylz2-mCherry made by inserting a promoter of zebrafish mylz2 gene and mCherry coding sequence into a pminiTol2 vector,and pmini-Chordin-mCherry containing a promoter sequence of Chordin gene from Branchiostoma floridae instead The two assembled vectors were co-injected with in vitro synthesized Tol2 transposase mRNA into unfertilized eggs of amphioxus B.floridae respectively.After injection and fertilization,the injected embryos emitted conspicuous red fluorescent protein(RFP)signal and were cultivated into adults(F0).Then,they were mated with wild-type(WT)adults to generate transgenic offspring.The results showed that about 30%of F0 individuals produced offspring emitting RFP signal,with transmission ratios up to 35%,indicating that Toll system is highly efficient in amphioxus model.Whole-mount in situ hybridization(WISH)and laser confocal microscopy revealed that mCherry was faithfully expressed in muscular tissues including somites,notochord,primary gill slits,and club-shaped gland in mylz2-mCherry transgenic embryos or larvae.Thus,the presentmylz2-mCherry transgenic line is a commendable model for studying the development of amphioxus muscle as well as the origin and evolution of vertebrate muscular tissues.However,in Chordin-mCherry transgenic embryos,mCherry expression pattern essentially followed that of endogenous Chordin gene,and specifically appeared in dorsal blastopore lip at gastrula stages as well as dorsal axial mesoderm and neural floorplate at neurula stages,but no RFP signal was detected in the adults.Such a transgenic line is decent candidate for marking notochord development and studying transcriptional regulation of endogenous Chordin gene.Moreover,RFP expression was observed in atrial cavity,velum and notochord in mylz2-mCherry transgenic adults although the expression pattern varied among individuals.Southern blot revealed that those expression variations were caused by distinct integration sites of mylz2-mCherry fragment,and inverse PCR also confirmed different insertion sites,and most insertions were mediated byTol2 system.Thus,we successfully developed an efficient Tol2-mediated transgenic method for amphioxus and obtained two transgenic amphioxus lines for the first time in this study.