Effects Of STAT1 Gene On Ibdv And Ndv Proliferation In Chicken DF-1 Cells

The production of viral vaccines with cell matrices is safe and efficient,and commonly used cells such as DF-1 and Vero play a huge role in vaccine production as matrices.However,due to cellular immunity,many types of viruses cannot be amplified in these cells,or the amplification efficiency is low.With the in-depth study of genes related to immune signaling pathways,gene editing technology can be used to knock out endogenous immune genes in cells or animals,or insert exogenous immune genes,which can fundamentally change cellular immune traits,and then optimize the vaccine production process and improve the vaccine production efficiency.The key role of STAT1 gene in signal transduction and transcriptional activation can affect the function of immune signaling pathway.In this study,we knocked out the STAT1 gene in chicken DF-1 cells to explore the effect of STAT1-ko cells on the proliferation of infectious bursal disease virus（IBDV）and Newcastle disease virus（NDV）viruses.The results of the study are as follows:1.Knockout of STAT1 gene in chicken DF-1 cells by CRISPR/Cas9technology.In order to obtain a chicken DF-1 cell line with a stable knockout of the STAT1 gene,three sg RNA targeting the second exon of STAT1 were designed,and the sg RNA knockout vector was co-transfected into chicken DF-1 cells.The transfected cells clones were collected by manual picking and expanded for PCR identification and sequencing analysis.The results showed that fragment-shift deletions were obtained in the target site of the STAT1 gene,indicating that a chicken DF-1 cell line with stable STAT1 knockout was derived.In order to verify the effect of knockout of STAT1 gene on the growth rate and cell morphology of chicken DF-1 cells,the morphology the wild type and knockout cells was examined under a microscope;then the growth curves of the two types of cells were drawn;lastly the expression levels of genes related to cell growth and adhesion were examined by quantitative PCR.The results showed that the growth rate and cell morphology of STAT1-ko cells were similar to wild type cells,the expression levels of cell growth and adhesion related genes were not significantly different,indicating that knocking out the STAT1 gene did not affect the growth rate and cell morphology of chicken DF-1 cells.2.Effect of knockout of STAT1 gene on proliferation of IBDV in DF-1 cells.STAT1-ko and wild type DF-1 cells were infected with IBDV,followed by q-PCR and TCID₅₀ assays,to examined the effect of STAT1 gene on viral proliferation in chicken DF-1.The results showed that,compared with wild type DF-1 cells,the expression of immune-related genes OASL was significantly reduced in STAT1-ko cells,while the viral gene expression and titer of IBDV virus were significantly increased.These results,indicate that STAT1-ko cells were more permissive to IBDV.3.Effects of Knockout of STAT1 Gene on NDV Infection of Chicken DF-1cells.STAT1-ko and wild type DF-1 cells were infected with NDV virus,followed by q-PCR and TCID₅₀ assays,to examined the effect of knocking out STAT1 gene on viral proliferation in chicken DF-1.The results showed that,compared with wild type DF-1 cells,the expression levels of STAT1 downstream immune-related genes OASL was significantly reduced and interferon and other immune genes in STAT1-ko cells,while the expression levels of NDV genes and virus titers were significantly increased.These results,indicate that STAT1-ko cells more permissive to NDV virus.In summary,stable STAT1-ko chicken DF-1 cells line were successfully established by using CRISPR/Cas technology.And the proliferation rate of IBDV and NDV viruses in STAT1-deficient chicken DF-1 cells was increased.The results provide an important reference for the follow-up study of chicken STAT1gene and the follow-up application of chicken DF-1 cells.