Efficient <i>PRNP</i> deletion in bovine genome using gene-editing technologies in bovine cells
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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<i>PRNP</i>基因编码的朊病毒（prion）相关疾病，如牛海绵状脑病（bovine spongiform encephalopathy, BSE，即疯牛病），是牛的致命性神经退行性疾病，但由于缺乏适配的细胞系或突变模型，通过基因缺失开展此类疾病的研究受到了极大限制。 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细胞用于相关研究的永生化成纤维细胞系。为此，本研究设计开展三项核心内容：1）通过导入<i>Bmi-1</i>与<i>hTert</i>基因，探究原代成纤维细胞的永生化诱导方法；2）在上述永生化细胞中开展<i>PRNP</i>基因的敲除研究；3）利用基因敲除（knockout, KO）细胞系评估基因表达情况与胚胎发育潜能。 本研究以一头雄性新生牛的原代细胞为材料，通过导入<i>Bmi-1</i>与<i>hTert</i>基因成功实现细胞永生化。该永生化细胞系连续培养超过180天，其倍增时间与细胞形态未发生任何异常改变。 此外，为敲除<i>PRNP</i>基因，本研究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酶切实验与测序验证，成功获得携带<i>PRNP</i>突变的克隆细胞系。 本研究选取4株<i>PRNP</i>基因敲除细胞系用于体细胞细胞核移植（somatic cell nuclear transfer, SCNT），重构胚胎可发育至囊胚阶段。在永生化细胞中敲除<i>PRNP</i>后，<i>CSNK2A1、FAM64A、MPG</i>与<i>PRND</i>基因的表达受到显著调控。 综上，本研究通过导入<i>Bmi-1</i>与<i>hTert</i>基因成功构建了牛永生化成纤维细胞系，并利用TALEN技术在该细胞系中实现了<i>PRNP</i>基因的高效敲除。该高效<i>PRNP</i>基因敲除技术有望为加深对牛体外（in vitro）朊病毒蛋白功能的理解提供有力的研究工具。
