利用重组慢病毒载体共表达Cas9-sgRNA构建TLR4基因敲除DF1细胞系

Construction of TLR4 gene knockout DF1 cell line by co-expression of Cas9-sgRNA with recombinant lentiviral vector

Toll样受体4（TLR4）是TLRs家族成员之一,可识别革兰阴性菌细胞壁脂多糖（LPS）和某些病毒蛋白,进而激活天然免疫应答,在细菌和病毒感染性疾病的发生过程中起重要作用。本研究靶向禽源TLR4基因第一外显子,设计、构建了2个sg RNA的CRISPR/Cas9双质粒表达系统,转染禽DF1细胞。流式细胞仪分选、测序验证了在家禽DF1细胞系成功敲除了TLR4基因。然后利用GV393慢病毒表达载体,构建了CAG启动子启动Cas9的共表达载体Lv-CAG-Cas9-U6-sg RNA。通过转染、流式分选、培养单细胞克隆、靶基因组测序以及Western-blot检测,筛选获得1株TLR4基因敲除的DF1细胞系。结果表明,TLR4基因敲除细胞系与野生型细胞生长动力学无显著差异。本研究验证了共表达Cas9和sg RNA可以在禽源细胞系实现基因编辑,为利用重组慢病毒共表达Cas9和sg RNA策略探索构建敲除鸡模型研究奠定了理论基础和科学依据,也为研究禽TLR4基因功能、家禽天然免疫应答机制提供了细胞模型。

Toll-like receptor 4（TLR4）,a member of the TLRs family,recognizes Gram-negative bacterial cell lipopolysaccharide（LPS） and certain viral proteins,and activates the innate immune response.It plays an important role in bacterial and viral infection.In the current study,two sg RNAs recombinant plasmid targeting the first exon of TLR4 was designed and constructed,co-transfected with Cas9 plasmid into DF1 cells.The transfected cells were sorted by flow cytometry.TLR4 knockout chicken DF1 cell line was confirmed by sequencing.Then the GV393 lentiviral expression vector was used as the backbone to construct the Lv-CAG-Cas9-U6-sg RNA.DF1 cell line with TLR4 knocked-out were screened by transfection,flow cytometry sorting,single cell cloning,sequencing and Western-blot methods.In result,TLR4 knockout cell line exhibited similar growth characteristics with wild type cells.It has demonstrated that co-expression of Cas9 and sg RNA could be used to perform gene editing in avian cell lines.The findings herein provide a theoretical basis for constructing the model of knockout chicken through co-expression of Cas9 and sg RNA using recombinant lentivirus and provide a cellular model for studying the function of avian TLR4 gene and the mechanism of innate immune response in chicken.