We developed knock-in vector system of human Noggin mature sequence with glutathione S-transferase (GST) containing factor Xa protease linker to facilitate the subsequent purification of recombinant protein. To achieve this, bovine ear fibroblast cells were isolated and transfection conditions were optimized by electroporation. To generate knock-in vector, human Noggin lacking its native signal peptide is fused to GST and foot and month disease virus 2A (F2A), and then inserted into bovine β-casein gene exon 3. We also generated enhanced green fluorescent protein (EGFP) expression vector of GST-human Noggin mature fused to β-casein signal peptide and F2A, and successfully detected recombinant human Noggin protein secreted into culture media, followed by factor Xa cleavage. Then, we co-transfected human Noggin knock-in vector with single-guided RNA and Cas9 expression vectors into bovine ear fibroblasts and obtained the stably-integrated colonies by antibiotic selection. PCR screening analysis revealed that 26 out of 35 colonies positively integrated human Noggin knock-in vector into bovine β-casein locus. One of positive clones was subjected to chromosome analysis, presenting normal karyotypes. Our data may provide the additional purification guideline of recombinant proteins by tagging GST with a protease linker sequence in the upstream of target genes and a high efficiency of integration ratio into bovine β-casein locus.

中文翻译：

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牛耳成纤维细胞β-酪蛋白基因位点中带有GST标签的人Noggin的CRISPR / Cas9敲入

我们开发了人Noggin成熟序列的敲入载体系统，该载体系统包含含有因子Xa蛋白酶接头的谷胱甘肽S-转移酶（GST），以利于后续纯化重组蛋白。为此，分离牛耳成纤维细胞并通过电穿孔优化转染条件。为了生成敲入载体，将缺乏其天然信号肽的人Noggin融合到GST和足月病病毒2A（F2A），然后插入牛β-酪蛋白基因外显子3。我们还产生了增强的绿色荧光蛋白（EGFP ）与β-酪蛋白信号肽和F2A融合的成熟GST-人Noggin表达载体，成功检测到分泌到培养基中的重组人Noggin蛋白，然后进行Xa因子切割。然后，我们将人Noggin敲入载体与单引导RNA和Cas9表达载体共转染到牛耳成纤维细胞中，并通过抗生素选择获得了稳定整合的菌落。PCR筛选分析显示，在35个菌落中，有26个将人Noggin基因敲入载体阳性整合到牛β-酪蛋白基因座中。对阳性克隆之一进行染色体分析，显示出正常的核型。我们的数据可以通过在靶基因上游用蛋白酶接头序列标记GST以及在牛β-酪蛋白基因座中的高整合率来为重组蛋白提供额外的纯化指导。PCR筛选分析显示，在35个菌落中，有26个将人Noggin基因敲入载体阳性整合到牛β-酪蛋白基因座中。对阳性克隆之一进行染色体分析，显示出正常的核型。我们的数据可以通过在靶基因上游用蛋白酶接头序列标记GST以及在牛β-酪蛋白基因座中的高整合率来为重组蛋白提供额外的纯化指导。PCR筛选分析显示，在35个菌落中，有26个将人Noggin基因敲入载体阳性整合到牛β-酪蛋白基因座中。对阳性克隆之一进行染色体分析，显示出正常的核型。我们的数据可以通过在靶基因上游用蛋白酶接头序列标记GST以及在牛β-酪蛋白基因座中的高整合率来为重组蛋白提供额外的纯化指导。