Efficient <i>PRNP</i> deletion in bovine genome using gene-editing technologies in bovine cells

Even though prion (encoded by the <i>PRNP</i> gene) diseases like bovine spongiform encephalopathy (BSE) are fatal neurodegenerative diseases in cattle, their study via gene deletion has been limited due to the absence of cell lines or mutant models. In this study, we aim to develop an immortalized fibroblast cell line in which genome-engineering technology can be readily applied to create gene-modified clones for studies. To this end, this study is designed to 1) investigate the induction of primary fibroblasts to immortalization by introducing <i>Bmi-1</i> and <i>hTert</i> genes; 2) investigate the disruption of the <i>PRNP</i> in those cells; and 3) evaluate the gene expression and embryonic development using knockout (KO) cell lines. Primary cells from a male neonate were immortalized with <i>Bmi-1</i>and <i>hTert</i>. Immortalized cells were cultured for more than 180 days without any changes in their doubling time and morphology. Furthermore, to knockout the <i>PRNP</i> gene, plasmids that encode transcription activator-like effector nuclease (TALEN) pairs were transfected into the cells, and transfected single cells were propagated. Mutated clonal cell lines were confirmed by T7 endonuclease I assay and sequencing. Four knockout cell lines were used for somatic cell nuclear transfer (SCNT), and the resulting embryos were developed to the blastocyst stage. The genes (<i>CSNK2A1, FAM64A, MPG and PRND</i>) were affected after <i>PRNP</i> disruption in immortalized cells. In conclusion, we established immortalized cattle fibroblasts using <i>Bmi-1</i> and <i>hTert</i> genes, and used TALENs to knockout the <i>PRNP</i> gene in these immortalized cells. The efficient <i>PRNP</i> KO is expected to be a useful technology to develop our understanding of <i>in vitro</i> prion protein functions in cattle.尽管由PRNP基因（PRNP gene）编码的朊病毒（prion）疾病，如牛海绵状脑病（bovine spongiform encephalopathy, BSE），属于牛的致命性神经退行性疾病，但由于缺乏相应细胞系或突变模型，相关基因敲除研究始终受到限制。本研究旨在构建一种可便捷应用基因组工程技术以制备基因修饰克隆供研究使用的永生化成纤维细胞系。为此，本研究设计了三项研究内容：1）通过导入Bmi-1与hTert基因，诱导原代成纤维细胞永生化；2）在上述永生化细胞中敲除PRNP基因；3）利用敲除（knockout, KO）细胞系评估基因表达与胚胎发育情况。研究人员从一头雄性新生牛体内分离原代细胞，通过导入Bmi-1与hTert基因实现细胞永生化。该永生化细胞可连续培养超过180天，其倍增时间与细胞形态均未发生明显变化。进一步地，为敲除PRNP基因，研究人员将编码转录激活因子样效应物核酸酶（transcription activator-like effector nuclease, TALEN）对的质粒转染至细胞中，并对转染后的单细胞进行扩增培养。通过T7核酸内切酶I检测法与测序验证，成功获得携带目标突变的克隆细胞系。选取4株PRNP敲除细胞系用于体细胞核移植（somatic cell nuclear transfer, SCNT），重构胚胎可发育至囊胚期。在永生化细胞中，PRNP基因敲除后会影响CSNK2A1、FAM64A、MPG及PRND这4个基因的表达。综上，本研究利用Bmi-1与hTert基因成功构建了牛永生化成纤维细胞系，并通过TALEN技术在该细胞系中实现了PRNP基因的高效敲除。该高效PRNP基因敲除技术有望为阐明牛朊病毒蛋白的体外（in vitro）功能提供有力的研究工具。