Effect Of Scriptaid On In Vitro Growth Of APN Gene-edited Pig Skin Fibroblasts And Early Development Of Re-cloned Embryos

In recent years,somatic cell nuclear transfer(Somatic cell nuclear transfer,SCNT)technology has shown great application value in the creation of genetically modified animals.It is a kind of technology with broad application prospect in livestock disease resistance breeding material creation,disease model construction and basic medical research.It has been proved feasible to reclone SCNT,with somatic cells of transgenic cloned animals as nuclear donors to obtain more transgenic animals.However,there are few studies on re-cloning of transgenic pigs,and the efficiency of re-cloning is not ideal.Previous studies have shown that the treatment of non-transgenic donor cells or SCNT reconstructed embryos with histone deacetylation inhibitor Scriptaid can improve the development of cloned embryos.We previously obtained APN gene editing clones of porcine transmissible gastroenteritis virus(Porcine transmissible gastroenteritis virus,TGEV).In order to establish and optimize the preparation of APN gene editing cloned porcine somatic cells and the production scheme of recloned embryos,and to provide technical reference for the realization of APN gene editing pig population expansion in the future,In this study,a skin fibroblast cell line was isolated from porcine tissue edited by APN gene and the possibility of its re-cloning was discussed.then somatic cells or embryos were treated with Scriptaid.The effects of Scriptaid on the growth of somatic cells in vitro and the early development of re-cloned embryos were also studied.The specific results are as follows:(1)Etablishment and re-cloning of APN porcine skin fibroblasts.The skin fibroblast cell line of APN pig was successfully obtained by culture in vitro,which has the biological characteristics of general fibroblasts.Compared the cleavage rate,the blastocyst rate,the total number of blastocyst with ungenetically edited porcine skin fibroblasts,there was no significant difference(78.8%;3.56;28 Vs.76.24%;4.11%;34,,P>0.05).(2)Effects of Scriptaid treatment on APN pig skin fibroblasts and their re-cloning.It was found that 500nM Scriptaid treatment for 24 h inhibited cell growth,but had no significant effect on cell survival and morphology.Compared the cleavage rate,the blastocyst rate,the total number of blastocyst with untreated group,there was no significant difference(73.99%;4.41%;25 Vs.78.77%;2.7%;40,P>0.05).(3)Effect of Scriptaid on the development of SCNT embryos of APN pigs in vitro.The cleavage rate,blastocyst rate and total blastocyst cell number of SCNT embryos in Scr iptaid treated group were significantly better than those in untreated group when SCNT em bryos were cloned from SCNT embryos treated with 500 nM Scriptaid for 14-16 h(80.55%;5.8%;32 Vs.50.93%;3.1%;30,P<0.05).In conclusion,the skin fibroblast cell line of APN gene editing cloned pig was success fully established,and the treatment scheme which could promote the development of reclo ned embryo was established based on Scriptaid intervention.Even if APN porcine skin fibr oblasts of 3 x 6 passages were used to produce recloned embryos,the reconstructed embry os were treated with Scriptaid for 14-16 h,and the developmental efficiency of the recons tructed embryos could be improved.In conclusion,this study confirmed the feasibility of APN porcine somatic cell line establishment and its recloning,and laid a solid foundation f or the future work of APN pig re-cloning against porcine transmissible gastroenteritis.