Introduction:Marker-free transgenic technology is highly desired to create genetically modified pigs,in term of food safetyand consumer ethics.AΦC31 integrase has been used to introduce site-specific transgene integr...
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Introduction:Marker-free transgenic technology is highly desired to create genetically modified pigs,in term of food safetyand consumer ethics.AΦC31 integrase has been used to introduce site-specific transgene integration into pseudo attPsites of animal *** represents a powerful tool to produce transgenic *** we attempted to examineits efficacy to mediate transgene integration into pig *** also wished to establish a marker-free transgenictechnology in large farm *** and methods:Donor and acceptor pigs were Hubei White Pig raised in the farm of Institute of Animal Science and VeterinaryMedicine,Hubei Academy of Agricultural ***+and PCMV-1NT were gifts of ***(StanfordUniversity).PEGFP-N1-attB reporter plasmid was constructed in our group.ΦC31 mRNA were purchased fromStanfordbio Inc.(Jiangsu).Results and discussion:ΦC31 mRNA and attB-containing pEGFP-N1 plasmid(PEGFP-N1-attB)were microinjected into 120 pig 1-cellzygotes and transferred into 5 surrogate pigs.32 piglets were live born from 4 acceptor *** analysis showed that 7 of them were attB and EGFP positive *** transgenic positiverate is 7/32=22％;gene transfer efficiency is 7/120=5.83%.In comparison to naked DNA pronuclearmicroinjection,this efficiency is significantly *** hot spot pseudo attP site (pig pseudo attP-1,ppp-1)was identified from2 piglets by ***-1 is located in an intergenic region of pig chromosome 1,implyingthat transgene integration into this site does not destroy any endogenous *** to now no abnormal phenotypeswere observed in term of birth weight,animal *** also demonstrated that PPP-1 appears to be safeharbor for transgene integration.
口蹄疫(Foot and Mouth Disease,FMD)是一种急性、热性、高度接触性传染病,主要感染偶蹄目动物,因其感染性极强,传播速度快,造成的经济损失巨大,被国际兽疫局列为A类传染病,该疾病主要是由口蹄疫病毒(Foot and Mouth Disease Virus,FMDV...
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口蹄疫(Foot and Mouth Disease,FMD)是一种急性、热性、高度接触性传染病,主要感染偶蹄目动物,因其感染性极强,传播速度快,造成的经济损失巨大,被国际兽疫局列为A类传染病,该疾病主要是由口蹄疫病毒(Foot and Mouth Disease Virus,FMDV)引起的.本文针对口蹄疫病毒基因组保守区3D,使用RNAi技术与转基因技术相结合,培育出抗口蹄疫转基因羊新品种.本实验筛选出了针对口蹄疫病毒3D序列的shRNA，双荧光素酶报告系统检测出抑制效率高达93.97％。随后，通过原核显微注射获得抗口蹄疫转基因山羊，获得61只微注射山羊，经PCR筛选和Southern Blot鉴定得到7只转基因阳性个体，又通过Northern Blot和qPCR对表达出的外源siRNA进行定性分析及定量检测，结果证实制备的转基因山羊上皮细胞能够稳定表达干扰口蹄疫病毒3D序列的siRNA，且表达量存在差异。
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