Nucleofection, an efficient nonviral method to transfer genes into human hematopoietic stem and progenitor cells
Nucleofection, an efficient nonviral method to transfer genes into human hematopoietic stem and progenitor cells
复制标题
DOI：
10.1089/scd.2006.15.278
复制
发表时间：
2006-04-01
影响因子：
4
通讯作者：
Giebel, B
中科院分区：
文献类型：
--
作者：
Von Levetzow, G;Spanholtz, J;Giebel, B
对单细胞以及完整生物体的遗传程序进行有针对性的操作极大地增强了我们对细胞和发育过程的理解，也应该有助于增加我们对初级人类干细胞（例如干细胞）的了解。例如，造血干细胞（HSC），在未来几年内。这种遗传方法的一个基本要求是存在一种可靠且有效的方法将遗传元件引入活细胞中。逆转录病毒和慢病毒技术可有效转导原代人类造血干细胞，但仍然费力费时，并且需要特殊的安全条件，而许多实验室并不具备这些条件。在我们的研究中，我们优化了核转染技术，一种改进的电穿孔策略，将质粒DNA引入新鲜分离的富含HSC的人CD34(+)细胞中。使用增强型绿色荧光蛋白 (eGFP) 编码质粒，我们获得了约 80% 的转染效率和 50% 的平均存活率。使用 GFU-GEMM 和长期培养起始细胞 (LTC-IC) 进行功能测定，我们证明除了存活率降低外，核转染方法本身不会明显改变原始造血细胞的短期或长期细胞命运。因此，我们得出结论，核转染方法是对原始造血细胞进行遗传操作的可靠且有效的方法。
The targeted manipulation of the genetic program of single cells as well as of complete organisms has strongly enhanced our understanding of cellular and developmental processes and should also help to increase our knowledge of primary human stem cells, e. g., hematopoietic stem cells (HSCs), within the next few years. An essential requirement for such genetic approaches is the existence of a reliable and efficient method to introduce genetic elements into living cells. Retro- and lentiviral techniques are efficient in transducing primary human HSCs, but remain labor and time consuming and require special safety conditions, which do not exist in many laboratories. In our study, we have optimized the nucleofection technology, a modified electroporation strategy, to introduce plasmid DNA into freshly isolated human HSC-enriched CD34(+) cells. Using enhanced green fluorescent protein (eGFP)-encoding plasmids, we obtained transfection efficiencies of approximately 80% and a mean survival rate of 50%. Performing functional assays using GFU-GEMM and long-term culture initiating cells (LTC-IC), we demonstrate that apart from a reduction in the survival rate the nucleofection method itself does not recognizably change the short- or long-term cell fate of primitive hematopoietic cells. Therefore, we conclude, the nucleofection method is a reliable and efficient method to manipulate primitive hematopoietic cells genetically.