Investigating The Effection And Mechanism Of SIRT2 On The Efficiency Of Gene Transfection Into Bovine Somatic Cells

Gene transfection is important in gene function and animal cell biology research.However,an important limitation of gene transfection,specifically non-viral gene transfection,is that the target gene is not highly expressed.Gene transfection is a multistep process and inefficiencies at any stage result in a dramatic decrease in gene delivery.In the case of gene transfection,strategies are needed to increase and accelerate the migration of DNA through the cytoplasm and transport it through the nuclear membrane.Here we reported that SIRT2 downregulation significantly improved gene transfection efficiency compared with the control group.In order to study the mechanism,we constructed SIRT2 expression vector pEGFP-SIRT2 and analyzed the intracellular microtubule acetylation level by western blot.The results showed that the microtubule acetylation was decreased by SIRT2 overexpression compared with the negative control group,while the acetylation level of the SIRT2-inhibited group(SiRNA-2)was significantly higher.The expression of SIRT2-EGFP was detected by fluorescence microscopy.And the results showed that SIRT2 was co-located with microtubules.We also found that SIRT2 inhibition failed to significantly increase TERT expression in TERT-bBMMs.This finding suggested that SIRT2 inhibition possibly resulted in increased transfection efficiency by increasing plasmid trafficking.In addition,The evidence of effect of SIRT2 inhibition on developmental potential of embryos confirmed that SIRT2 inhibition did not affect the developmental potential of SCNT embryos.Macrophages,as the forefront of innate immune defense,have an important role in the host responses to mycobacterial infection.Therefore,a stable macrophage cell line is needed for future bovine immune system research on the bacterial infection.In this study,we established a bovine macrophage cell line by introducing the human telomerase reverse transcriptase(hTERT)gene into bovine bone marrow-derived macrophages(bBMMs).The TERT-bBMMs cells expressed macrophage surface antigen(CD11b,CD282)and upregulated expression of the cytokines IL-1?,IL-6,IL-10,IL-12,TNF-? in response to bacterial invasion.These results demonstrate that this cell line provide reliable cell model system for future studies on interactions between the bovine macrophages and Mycobacterium tuberculosis.In summary,SIRT2 inhibition increased the transgene expression.However,the increased expression was not attributed to the events that occurred when the plasmid entered the nucleus.SIRT2 inhibition possibly increased the transfection efficiency by promoting plasmid trafficking.SIRT2 inhibition did not affect the developmental potential of SCNT embryos.Therefore,enhanced gene transfection by using SIRT2 inhibition could be a feasible strategy.