Construction Of Inducible Sohlh1 Gene Knockout Cell Line With CRISPR/Cas9 System On Pig

It is reported that Sohlh1 gene knockout leads to male sterility and female infertility in mice, and it has no related reports on pig. The CRISPR/Cas9 RNA-guided nucleases system is a transformative technology for biology, and it can cleave specific DNA target sites in living cells and organisms. This research adopts the Tet-on system and CRISPR/Cas9 technology to establish the inducible knockout pig's Sohlh1 gene cell line, which provides the basis for the production of knockout clone pig model, and researches the effect on the reproduction and development of pigs.This research has optimized Cas9 gene and reconstructed plv-sg RNA vector at first;,Through lentiviral PCW-e Sp Cas9(1.1) vector packaging, then infecting pigs fetal fibroblasts and drug selecting; Through sequence analysing of pig Sohlh1 gene CDs area, we predicted Sohlh1 gene Cas9 targets and constructed U6-sg RNA instantaneous expression element, then transiently transfected cells with stably integrated Cas9 gene and analysed the knockout activity of each target site. Lentiviral plv-sg RNA-2A-GFP specificity vector concentrated and infected pig fetal fibroblasts with e Sp Cas9(1.1) gene transfection, and then screening positive cells by drug and fluorescence detection at last.Results of the trial indicated that(1) by sequencing and fluorescence detection, optimization and transformation of low off-target rate of PCW-e Sp Cas9(1.1) vector support and with fluorescent-labeled plv-sg RNA-2A-GFP vector successfully;(2) The positive cells, which were transfected with lentiviral PCW-e Sp Cas9(1.1) vector, induced by Dox and the expression of Cas9 was detected;(3) Sohlh1 gene CDs area sequence is basically in line with the data reported in NCBI by PCR amplification and sequencing;(4) It can be seen that the target point of 3, 4 and 6 have have a knockout activity, while the 5 have no or low, by transient transfection experiments, and then we choose to build sg RNA-specific lentiviral vector with the target point of 3 and 4;(5) The 293 FT cells expressed GFP upon transfection with sg RNA-specific lentiviral vector, so lentiviral vector was packaged successfully;(6) The e Sp Cas9(1.1)-positive cells were infected with concentrated sg RNA lentiviral, and then they were induced by Dox, finding that Sohlh1 gene knockout by sequencing and analyzing. Therefore, we can come to the conclusion that the inducible knockout Sohlh1 gene cell line was constructed successfully.In summary, the study obtained the inducible Sohlh1 gene knockout cell line which is meaningful for the production of gene knockout clone pig and the research of reproductive disease,and it is also very useful for further study of Sohlh1 regulatory mechanism and function on reproduction development.