TALENs-mediated Study Of Goat BLG Gene Knockout And HLF Gene Knockout

TALENs gene targeted modification technology has developed rapidly in recent years, which can easily and efficiently produce a variety of effects of mutations in the target site of the target gene, this technology has been applied to a variety of plant and animal cells and individuals modification studies.Compared with the classical gene targeting, TALEN nuclease under the joint action with the gene targeting vector having a foreign gene knockout and knock-point advantage, "one step" to complete the target gene. In this study, by using gene editing technology TALENs goat BLG gene knockout and knock-in knock-out position designated human lactoferrin hLF exogenous gene, people sensitized β-lactoglobulin in goat milk is substituted human lactoferrin, and thereby improve the nutritional value of goat’s milk consumption.The purpose of this study was to TALENS technology and goat β-lactoglobulin regulatory sequence, PCR amplification of cDNA obtained hLF gene sequence, the human cytomegalovirus promoter/enhancer (Pcmv) and SV40 poly A and HSV-TK negative screening genes and other components to construct the breast screening containing neomycin resistance gene (Neo) specific expression of hLF gene targeting vector.TALENS and breast-specific targeting vector was constructed to obtain transfected goat fetal fibroblasts, genetically screened monoclonal cell line, and prepared on the BLG gene knock-point hLF transgenic goats by somatic cell nuclear transfer technology. Transplant recipients and pregnant transgenic goat born, to nurture a new generation of high yield and quality without people sensitized, and rich in beneficial to humans human lactoferrin transgenic dairy goat milk new line.Goat β-lactoglobulin gene (BLG) is located on chromosome 11, the full-length 8088bp, encoding BLG transcription unit length 4698bp,5’untranslated region of 2148bp,3’untranslated region of 1242bp, containing seven exons, six introns. The study was designed experiment, it has been reported in accordance with the TALENs design principles to goat BLG gene sequence as a template design TALENs, in the first exon of ATG-2189 near the start codon selected two target sequences, as a guide to construct expression vectors TALENs the target recognition sequence. Select the target sequence corresponding to residues dual recognition module and backbone vector, after ligation, transformation, screening, identification, and ultimately get two pairs TALEN expression vector (TALEN-L12、TALEN-R12, TALEN-L18/ TALEN-R18), TALENs expression vector is TALENs activity identification and BLG gene designated modification research foundation.In order to be able to get support in goat mammary gland-specific and efficient expression of human lactoferrin in the present study was to goat BLG gene as a template, PCR were amplified goats 5’regulatory sequences 1035 bp and 3’regulatory sequences 1826 bp, and then to the present laboratory saved BLC14 basis vectors include the 5’regulatory sequence, CMV start/enhancer, hLF gene sequence cDNA, SV40polyA start/enhancer, NEO gene and 3’regulatory sequences, obtained by PCR amplification of the gene whose function method sequence, and then connecting the BLG control sequences, identify forward and reverse insertion sequences, picked the right carrier.After determining the correct mammary-specific gene expression vector was linearized by digestion, and then save the laboratory containing a negative selection gene sequence of HSV-TK is connected to obtain the present experiments require breast hLF gene-specific expression targeting vector, named BLTF-7-82.Gene targeting vector BLTF-7-82 and TALEN-L18/R18 plasmids were co-transfected goat fetal fibroblast cells by G418 and GCV drugs were screened for 7-14 days, to pick up cell growth and cell culture plate clone, ultimately, drug resistance fetal fibroblasts 125 were by PCR to obtain cell lines, gene targeting efficiency was 1.5 × 10-6,LDK-16 were used as donor cells somatic cell nuclear transfer to produce 151 reconstructed embryos reconstructed embryo transplant recipient goats 10,30 days after embryo transfer pregnancy is detected B-4 receptor (pregnancy rate 70%).