Objective: To investigate the proliferation of mouse splenic lymphocytes after shRNA mediated BTLA gene silence.
Methods: Three specific shRNAs and one nonspecific shRNA (scrambled) were ligated to pSilencer3.1-H1-neo plasmid and then subcloned into lentiviral vector pLB. Recombinant viral particles were harvested post-transduction to 293T cells. Mouse lymphocytes were infected with viral supernatant after 24 h incubation and continuously cultured till 4 days. Expression of eGFP was detected by fluorescence microscopy, efficiency of infection and expression of BTLA on lymphocyte cell by FCM. CCK-8 assay was used to detect the proliferation of lymphocytes.
Results: Lentiviral expression vectors pLB-shRNA/BTLA were successfully generated. The lentiviral particles were correctly packaged. Expression of BTLA protein in specific shRNA group was significantly decreased comparing to those in control group. O.D. value at A(450) of lymphocytes stimulated by anti-CD3 antibody showed significant difference compared with normal BTLA group (P < 0.05), while there was no difference between ConA stimulated group and control (P > 0.05).
Conclusion: Gene-specific shRNA can knockdown the expression of BTLA. The proliferation of lymphocytes stimulated by anti-CD3 antibody after RNAi demonstrates significant enhancement as compared to the unstimulated lymphocytes, while stimulated by ConA showed no difference compared to normal lymphocytes.