Abstract: Objective · To construct retroviral small guide RNA (sgRNA) expression vector for studying gene function in mouse T cells. Methods · The short hairpin RNA (shRNA)-expressing retroviral vector MSCV-LTR-miR30-PIG (LMP) was digested with Xho1 and Sal1 to obtain the backbone of the retroviral vector. The lentiviral sgRNA vector was used as template for PCR amplification to obtain the U6-sgRNA-PGK-Puro-BFP fragment. The PCR fragment was then assembled into the retroviral vector backbone by homologous recombination. To verify the effectiveness of the system, sgRNA sequences targeting Il17a, Rorc and Irf4 were designed and cloned. CD4 T cells isolated from Cas9 transgenic mice were infected with individual sgRNA retrovirus and differentiated into Th17 cells. After cytokine intracellular staining, the percentage of Il17a positive population was detected by flow cytometry. Unpaired t test was used to compare the data of independent samples between the two groups. Results · ①The sgRNA retroviral vector was successfully constructed. ② The retroviral vector was used to successfully deliver sgRNA to CD4 T cells, which mutated Il17a gene. ③The percentage of Il17a positive population was significantly reduced in the Rorc-sgRNA and Irf4-sgRNA positive populations (P<0.05). Conclusion · The sgRNA retroviral vector is successfully used to deliver sgRNA to target cells. In addition, the results of Rorc and Irf4 gene knockout experiments prove that the system can be used for studying gene function in mouse T cells.

Key words: CRISPR/Cas9, Cas9 transgenic mouse, retroviral vector, Th17 cell differentiation