Abstract:
Objective The Tet-on system was combined with the CRISPR/Cas9 system to establish a pig cell line with inducible Sohlh1 gene knockout.
Method Porcine fetal fibroblast (PFF) was isolated and cultured for infection with lentiviral vector PCW-eSpCas9(1.1), screened by 1 μg/mL purinomycin and induced by doxycycline (Dox). The pLV-sgRNA vector was modified and transfected with 293FT cells for validation. The pLV-sgRNA-2A-GFP specific vector containing Sohlh1 gene target was constructed for lentivirus packaging detection. PCW-eSpCas9(1.1) PFF cell was transfected by package virus infection and screened by 3 μg/mL blasticidin, followed by Dox induction for expression analysis and knockout efficiency detection.
Result PFF cell line, which was regulated by Dox to induce eSpCas9 (1.1) protein expression, was established. Without Dox addition, eSpCas9 (1.1) protein expression was not observed. Green fluorescence was expressed in 293FT cells, indicating the successful transformation of pLV-sgRNA-2A-GFP vector. The No. 3, 4 and 6 Sohlh1 gene targets had knockout activities. The pLV-sgRNA-2A-GFP specific vector was constructed with the two optimal targets, and the fluorescence expression showed that the vector lentivirus was successfully packaged. The PFF cell of stable PCW-eSpCas9(1.1) and pLV-sgRNA-2A-GFP transformation was constructed. After 72 hours of Dox induction, the knockout efficiency of Sohlh1 gene was 85%.
Conclusion The pig cell line with inducible Sohlh1 gene knockout is successfully constructed using the Tet-on system and CRISPR/Cas9 system, which lays a foundation for studying the function of Sohlh1 gene and preparing the model pig with conditional Sohlh1 gene knockout to remove germ cells.
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