Study On Competitive Inhibitors Of ID1 Molecules In Zebrafish(Danio Rerio)

Basic helix-loop-helix(bHLH)transcription factors play important roles in eukaryotic growth,development and cell differentiation.The bHLH transcription factor contains a bHLH domain consisting of a DNA-binding basic region and two alpha-helices domains linked by a loop.The differentiation inhibitor ID1 belongs to the bHLH protein family,which contains the HLH domain,but no basic domain.ID1 molecules can interact with b HLH protein,inhibit bHLH and DNA binding.Thus it is also known as DNA binding inhibitory factor.ET2 fusion protein fuses the E protein activation domain(AD)with the bHLH domain of SCL.ET2 fusion protein can be used as a competitive inhibitor of ID1.In this study,the tcf3 a,tcf3b,tcf12 and scla cDNA sequences of zebrafish were retrieved from GenBank.The tcf3 a,tcf3b,tcf12 and scla ORF were amplified,and ligated into pcDNA3.1(+)-c-myc vector after double restriction enzyme digestion.It was verified that pcDNA3.1(+)– tcf3a-c-myc,pcDNA3.1(+)– tcf3b-c-myc,pcDNA3.1(+)-tcf12-c-myc,pcDNA3.1(+)– scla-c-myc plasmids was constructed successfully by sequencing.The tcf3 a,tcf3b,tcf12 and scla ORFs were putatively translated into protein sequence and the domains were predicted.Therefore we can design the linker primers to construct plasmids coding fusion protein ET2.tcf3a-scla,tcf3b-scla,tcf12-scla,e2-2-scla,e2-2b-scla fusion amplicons inserted into pcDNA3.1(+)-c-HA vector repectively,pcDNA3.1(+)-tcf3b-scla-c-HA,pcDNA3.1(+)-e2-2a-scla-c-HA,pcDNA3.1(+)-tcf3a-scla-c-HA,PcDNA3.1(+)-e2-2b-scla-c-HA,pcDNA3.1(+)-tcf3a-scla-c-HA plasmids were successfully constructed.The constructed plasmids were used to analyze the inhibitory effect of five fusion proteins on ID1 in vitro transfection of EPC cells.The results showed that none of the five fusion proteins had significant inhibitory effect on ID1.In order to study the inhibitory effect of five fusion proteins on ID1 in vivo,we constructed the Tol2 transgenic plasmids expressing the fusion protein and fluorescent protein(Tag RFP)driven by heat shock protein 70 promoter(hsp70pro).These were repectively injected into zebrafisf embryos to construct the corresponding transgenic zebrafish(F0 generation).The sequencing results show that Tol2-hsp70pro-P2A-TagRFP,Tol2-hsp70pro-TCF3a-SCLa-P2A-Tag RFP,Tol2-hsp70pro-TCF3b-SCLa-P2A-TagRFP,Tol2-hsp70pro-TCF12-SCLa-P2A-Tag RFP,Tol2-hsp70pro-E2-2aSCLa-P2A-TagRFP,Tol2-hsp70pro-E2-2b-SCLa-P2A-TagRFP plasmids were constructed successfully.The corresponding transgenic zebrafish F0 generation was determined to be unsuccessful,and it is required.After that we need another re-injection in order to construct transgenic fish lines.