Study On Sheep Early Cloned Embryos Of Transfering Spidroin1Gene

The unapproachable biological characteristics of spider silk determines it has broad application in many fields. To obtain the active protein with spider silk characteristics using biological method, or animal fibers like spider silk using transgenic approach,is some scientific researchers’goal all these years.This study utilized the spider dragline silk protein gene4S、 pcDNA3.1and pIRES2-EGFP vector to contruct pcDNA3.1-4S and pIRES2-EGFP-4S expression vector,utilized the liposomal and linearized spider dragline silk protein gene4S to transfect sheep fibroblasts,then obtained the positive cell line;used the positive cell line as donor nuclear to obtain trans-Spidroinl gene sheep reconstruceted embryos by nuclear transfer; identified whether the the spider dragline silk protein gene integrated into the reconstruceted embryos.The results of this study were showed as follows.1. Construction of pcDNA3.1-4S and pIRES2-EGFP-4S expression vectorThe pcDNA3.1-4S expression vector was constructed by pcDNA3.1vector and spider dragline silk protein gene4S, the result showed that the spider dragline silk protein gene4S had integrated into the pcDNA3.1-4S expression vector after BglⅡ、BamHI double enzymes cutted and PCR identificated;the pIRES2-EGFP-4S vector was constructed by spider dragline silk protein gene4S and pIRES2-EGFP vector, the result showed that the spider dragline silk protein gene4S had integrated into the pIRES2-EGFP-4S vector after XhoⅠ, BglⅡ double enzymes cutted and PCR identificated.2. Establishment of trans-Spidroninl gene sheep fibroblasts cell lineUtilized the liposomal to transfect the sheep primary and passage fibroblast with0.4μg/mL linearized pcDNA3.1-4S and pIRES2-EGFP-4S,screened the sheep fibroblasts by culture media with400μg/mL G418and antibiotics, and finally obtained trans-pcDNA3.1-4S and pIRES2-EGFP-4S cell line; observed the morphology,drawed the growth curve,calculated the proliferation time of the transgene cell,all the results were accord to the normal fibroblast feature.3. Screened the method of sheep oocytes collectingCompared the effect of sheep oocytes collecting method by suction,cut and suction combined with cut, the results showed that the cut assisted with lineation can obtain about8oocytes (including A and B levels) each ovary.4. Vitro maturation of sheep oocytesUsed the media:TCM199-HCO3+2.2g/mL NaHCO3+5μg/mL FSH+1IU/mL LH+1μg/mL E2+50μg/mL Gentamicin+10%FBS+0.38mmol/L sodium pyruvate+10mmol/L Hepes as culture media to culture A and B levels of sheep oocytes,obtained the highest83.4%maturation rate,it showed that this culture media were fit for sheep oocytes vitro maturation.5. Parthenogenetic activation of sheep matured oocytesThe vitro matured sheep oocytes were firstly activated with5μmol IA23187,then cultured in2mmol/L6-DMAP for4h,it could obtain91.5%activation rate,after cultured84.7%of the parthenogenetic activation embryos were developed into morula. it showed that IA23187combined with6-DMAP were fit for parthenogenetic activation of matured sheep oocytes.6. Somatic cells nuclear transfer of transgene cellUsed the sheep fibroblasts and trans-spider dragline silk protein gene cell as the donor nuclear,after somatic cells nuclear transfer operation which were founded before,the results showed that the abnormal cell melt rate was72.7%,the melt rate of transgene cell was70.7%,there were no difference,it indicated that there were no influence of the exogenous gene integrated into the reconstruceted embryos on it’s development ability.7. Development ability of trans-spider dragline silk protein gene reconstruceted embryos by nuclear transferCultured the trans-spider dragline silk protein gene reconstruceted embryos by nuclear transfer,the morula rate of transgene cell as donor nuclear was11.6%,moreover the morula of abnormal cell as donor nuclear wasl3.8%,there were no difference between them transferred the trans-spider dragline silk protein gene reconstruceted embryos into the oviduct of the donor sheep,pregnance was observered,but no trans-spider dragline silk protein gene lamb obtained.In summary,this study used the pcDNA3.1-4S and pIRES2-EGFP-4S expression vector were constructed to obtain the transgene cell line;used the transgene cell as donor nuclear to obtain trans-spider dragline silk protein gene reconstruceted embryos, the reconstruceted embryos obtained had the ability developing into morula.this study built the foundation for further research of obtaining trans-spider dragline silk protein gene sheep in hair.