Knocking Out Sheep WNT2 And FGF5 Gene By CRISPR/Cas9-Mediated Gene Editing

Hair follicles(HF)is the most complex and distinctive mammalian micro organ control the sheep wool growth.The development of secondary HF is the mainly factor influence sheep wool quality,its growth depends on the successive and complex signal pathway interaction between dermis and epidermis.But only few research focus on the regular mechanism of the gene control SF development.Pervious research found WNT2 exist difference expression during induction period of SF morphogenesis.For further investigate WNT2 function and establish sheep gene editing technology method to optimize sheep wool quality.We use CRISPR/Cas9 system to knock out WNT2 on cell for validating WNT2 function work on gene control HF development.Then establish sheep FGF5 gene editing method based on Alpine Merino Sheep zygotes microinjection.The main result as follow.1.Culturing primary sheep skin fibroblast cell,designed and constructed WNT2 knock out vector pUC-sgRNA-pSpCas9(BB)-2A-Puro.Through lipofection to transfected cell and earned the WNT2 knock out cell validated by T7E1 enzyme digest detection.The edit efficiency is 14.35%.The Q-PCR find the expression of WNT2 is down-regulation extremely significant(P<0.01)and expression of CDH1 is up-regulation extremely significant(P<0.01).The expression of FGF5 and Caspase3 are up-regulation significant(P<0.05)show WNT2 inhabit FGF5 and activate apoptosis path when it down express.As the result,WNT2 is promote hair follicles growth and development.This will lay foundation for further investigate the molecular regulate mechanism of sheep HF morphogenesis.2.Comparison of different follicle stimulating hormones(FSH)dose,injection protocol and environment temperature during superovulation to optimize embryo recovered procedure of Alpine Merino sheep for zygote number every recovered treatment maximization.Find the best Alpine Merino Sheep superovulation procedure in winter is use six time decrease inject totally 10 mL Folltropin-V,each inject dose is 2.5mL,2mL,2mL,1.5mL,1mL,1mL at-8?.Recover zygotes at 40 h after donor first mating.To validate lamb through T7E1 enzyme digestion prove earn FGF5 knock out lamb.This result show successful established Alpine Merino sheep gene editing method and provide support for using gene editing technology to improve sheep wool quality.