Gonçalves Cristine, Gross Fabian, Guégan Philippe, Cheradame Hervé, Midou Patrick
Centre de Biophysique Moléculaire, CNRS, Orléans, France.
Biotechnol J. 2014 Nov;9(11):1380-8. doi: 10.1002/biot.201400324. Epub 2014 Oct 15.
Bioproduction of recombinant proteins (r-proteins) and recombinant lentiviral particles (r-lentiviral particles) requires robust transfections consisting of efficient protocols that are easy to implement, with good reproducibility for a maximum production of proteins and lentiviral particles in a short time with low cytotoxicity. This study evaluates the capacity of histidinylated polyethyleneimine I (PTG1) to facilitate robust DNA transfection, with low cytotoxicity, of Chinese hamster ovary (CHO) and human embryonic kidney (HEK293T) cells for the production of r-proteins and r-lentiviral particles. We report that PTG1 transfection of cells in suspension with a plasmid DNA encoding enhanced green fluorescent protein leads to 72 and 97% of transfected CHO and HEK293T cells respectively, and does not significantly affect cell viability. PTG1 transfection of 100 mL of CHO-S cell culture in suspension at a cell density of 2 × 10(6) cells /mL resulted in a high level of transfected cells and protein expression after transfection with 0.75 μg/mL plasmid DNA. Transfection with PTG1 is more efficient than LipofectAmine2000™, and gene expression is higher than observed with FreeStyle™ and JetPEI®. Tri-transfection of HEK293T packaging cells leads to the production of a higher level of r-lentiviral particles compared to the calcium phosphate method, and permits two harvests of viral particles within three days. These results show that PTG1 is a powerful new transfection reagent for cell lines frequently used for recombinant protein and lentiviral particle production. PTG1 could be used in protocols for bioproduction of therapeutic proteins such as antibodies for cancer treatments and viral vectors for gene therapy applications.
重组蛋白（r蛋白）和重组慢病毒颗粒（r慢病毒颗粒）的生物生产需要强大的转染技术，即要有易于实施的高效方案，在短时间内以低细胞毒性实现蛋白质和慢病毒颗粒的最大产量且具有良好的重复性。本研究评估了组氨酸化聚乙烯亚胺I（PTG1）促进对中国仓鼠卵巢（CHO）细胞和人胚肾（HEK293T）细胞进行低细胞毒性的强大DNA转染的能力，用于生产r蛋白和r慢病毒颗粒。我们报告称，用编码增强型绿色荧光蛋白的质粒DNA对悬浮细胞进行PTG1转染，分别导致72%和97%的CHO细胞和HEK293T细胞被转染，且对细胞活力没有显著影响。以2×10⁶个细胞/mL的细胞密度对100 mL悬浮的CHO-S细胞培养物进行PTG1转染，在用0.75 μg/mL质粒DNA转染后产生了高水平的转染细胞和蛋白质表达。用PTG1转染比LipofectAmine2000™更有效，基因表达高于使用FreeStyle™和JetPEI®时观察到的水平。与磷酸钙法相比，对HEK293T包装细胞进行三质粒共转染可产生更高水平的r慢病毒颗粒，并允许在三天内收获两次病毒颗粒。这些结果表明，PTG1是一种强大的新型转染试剂，适用于常用于重组蛋白和慢病毒颗粒生产的细胞系。PTG1可用于生物生产治疗性蛋白质（如用于癌症治疗的抗体）和用于基因治疗应用的病毒载体的方案中。
