Study Of TALEN-Mediated Site-Specified Integration LacS Gene With No Marker Cattle's Production

There is long history for livestock selection and breeding.The development of biology segnificantly promotes livestock breeding research.Compared with traditional gene transfer and gene targeting,gene editing tools(ZFN,TALEN and CRISPR)provide wide help for gene engeering research.The combination of somatic cell clone and gene editing technology make posibility for gene editing animal production.The most defect of common gene transfer is exogenous gene's random integration,which could give rise to endogenesis gene function change or exogenous gene mutation.Besides,the introduction of marker gene and anti-genes would be potential danger about bio-safity.Lactose intolerance is a widely phenomenon which could cause the gas and stomach pain.Lactase Sulfolobus Solfataricus is a type of lactase came from thermophilic archaebacteria,it's suitable reaction temprature is 75~100 ?.This study aimed to integrate LacS gene into diary cattle genome and expressed at breast by TALEN.Expect to produce marker-free transgene cattle,and provide a new way to solve lactose intolerance.The article's results are as follows.(1)A two-month old cow fetus was removed by cesarean section,and the skin of the body side was divested and cultured using tissue-piece inoculation.Obtained fibroblasts were analized by phenomenon and growth curve and used for LacS gene integration.(2)The ?-casein locus 2th intro was used as the target gene insert site and the LacS gene was integrated into the target site using mic rohomology-mediated end-joining.Two active TALENs,specific to the intergenic region,were designed to prevent the occurrence of potentially synergistic effects from neighboring genes.The activity of TALENs in fibroblasts was screened using the T7E1 assay and one is active.At the same time,the LacS gene vector which contains micro-homology arms was constructed.(3)A total of TALEN-encoding plasmids and LacS donor plasmid were transfected into P1 bovine fetal skin fibroblasts.The cultured single cells were transferred to 96-well cell culture plates to generate monoclones.Upon proliferation of passaged monoclones,the cells were transferred to 6-well culture plates and half of the associated cells were frozen.The remnant cells were used to isolate genomic DNA and perform PCR-mediated identification.In total,124 monoclones were generated.Only one clone was shown to be positive.The total knock-in rate was 0.8%.(4)The positive monoclone was used as a nuclear donor to generate reconstructed embryos with a cleavage rate of 66%(188/288).The efficiency of development to the blastocyst stage was 27%(51/188).Blastocyst and morula stage embryos were transfe rred to the uterus of synchronized estrus recipient heifers.The rate of pregnancy was 20%(4/20).