PDF(Pubmed)
Abstract：
We engineered HEK293T cells with a transgene encoding tetracycline-inducible expression of a Staphylococcus aureus nuclease incorporating a translocation signal. We adapted the unmodified and nuclease-engineered cell lines to grow in suspension in serum-free media, generating the HEK293TS and NuPro-2S cell lines, respectively. Transient transfection yielded 1.19 × 106 lentiviral transducing units per milliliter (TU/mL) from NuPro-2S cells and 1.45 × 106 TU/mL from HEK293TS cells. DNA ladder disappearance revealed medium-resident nuclease activity arising from NuPro-2S cells in a tetracycline-inducible manner. DNA impurity levels in lentiviral material arising from NuPro-2S and HEK293TS cells were undetectable by SYBR Safe agarose gel staining. Direct measurement by PicoGreen reagent revealed DNA to be present at 636 ng/mL in lentiviral material from HEK293TS cells, an impurity level reduced by 89% to 70 ng/mL in lentiviral material from NuPro-2S cells. This reduction was comparable to the 23 ng/mL achieved by treating HEK293TS-derived lentiviral material with 50 units/mL Benzonase.
摘要：
我们设计了HEK293T细胞，其转基因编码四环素诱导的金黄色葡萄球菌核酸酶的表达，并掺入了易位信号。我们调整了未修饰的和核酸酶工程化的细胞系，使其在无血清培养基中悬浮生长，产生HEK293TS和NuPro-2S细胞系，分别。瞬时转染从NuPro-2S细胞产生每毫升1.19×106个慢病毒转导单位(TU/mL),从HEK293TS细胞产生1.45×106TU/mL。DNA梯消失揭示了NuPro-2S细胞以四环素诱导的方式产生的中等驻留核酸酶活性。由NuPro-2S和HEK293TS细胞产生的慢病毒材料中的DNA杂质水平通过SYBRSafe琼脂糖凝胶染色检测不到。通过PicoGreen试剂直接测量显示DNA在来自HEK293TS细胞的慢病毒材料中以636ng/mL存在,来自NuPro-2S细胞的慢病毒材料中的杂质水平降低了89%至70ng/mL。这种减少与通过用50单位/mL的Benzonase处理HEK293TS衍生的慢病毒材料所实现的23ng/mL相当。
