Cas9-mediated Multiple Locus Gene Targeting For Bubalus Bubalis

Bubalus bubalis milk was famous for its high nutritional value, rich in calcium and energy, which is high quanlity milk. With the development of modern dairy industry, buffalo dairy industry, which is non-mainstream, is slowly stepped into the mainstream market.Chinese buffalo have huge amount of resources, but buffalo dairy industry started relatively late, with unbalanced regional dairy. Therefore, the development of buffalo dairy has great significance. Buffalo milk contain content of ?s1- casein which human milk does not exist,so, when human intake of Buffalo milk, they will be allergic. Our group has established a multiple locus gene targeting technology platform for a long time, taking advantage of ribosomal r DNA repeats, with transcriptionally active region as the target site, which can greatly improve the efficiency of gene targeting. This study selected CSN1S1 gene, whose expression is closely related to ?s1- of casein. Using multiple locus gene targeting technology and Cas9 artificial endonuclease, we knock-down the expression of CSN1S1.Firestly, We designed three RNAi target site For c DNA of CSN1S1 gene sequence by Biotechnology company, to construct sh RNA transiently expression plasmids that p GPU6-sh RNA-NEO-1,p GPU6-sh RNA-NEO-2,p GPU6-sh RNA-NEO-3, negative control plasmid p GPU6-sh RNA-NEO–NC. Then we through GFP eukaryotic expression vectors p EGFP-N1 and p EGFP-C2 to construct fusion expression plasmids p EGFP-N1-CSN1S1 and p EGFP-C2-CSN1S1.Thirdly, the expression of sh RNA vectors and the expression of green fluorescent fusion plasmids were co-transfected 293 T cells,which lead to the result that the expression of sh RNA vectors were all effective, the p GPU6-sh RNA-NEO-2,p GPU6-sh RNA-NEO-3 were slightly better than p GPU6-sh RNA-NEO-1.At the same time,we test the m RNA level of CSN1S1, the results also proved that the three targets were valid,but there was no significant difference between three targets.Then,we cloned the fragment U6-sh RNA-NEO of p GPU6-sh RNA-NEO-2 and p GPU6-sh RNA-NEO-3 through PCR, insert which into the buffalo universal targetingvector PUC19-HRDS1-HRDS2,so we get multiple points targeting plasmid PUC19-HRDS1-U6-sh RNA-NEO-HRDS2-1 and PUC19-HRDS1-U6-sh RNA-NEOHRDS2-2. The targeting plasmids and Cas9 expression plasmids were co-transfected into buffalo kidney fibroblasts, setting up three control groups,including the targeting plasmid transfected group, the targeting plasmids and the Cas9 expression plasmids co-transfected group and non-transfected group,which proved that the gene were inserted.