Programa de Carcinogênese Molecular, Coordenação de Pesquisa (CPQ), Instituto Nacional de Câncer (INCA), Rio de Janeiro, Brazil.
PLoS One. 2013;8(3):e60298. doi: 10.1371/journal.pone.0060298. Epub 2013 Mar 26.
Gene transfer to T lymphocytes has historically relied on retro and lentivirus, but recently transposon-based gene transfer is rising as a simpler and straight forward approach to achieve stable transgene expression. Transfer of expression cassettes to T lymphocytes remains challenging, being based mainly on commercial kits.
We herein report a convenient and affordable method based on in house made buffers, generic cuvettes and utilization of the widely available Lonza nucleofector II device to promote efficient gene transfer to T lymphocytes.
This approach renders high transgene expression levels in primary human T lymphocytes (mean 45%, 41-59%), the hard to transfect murine T cells (mean 38%, 36-42% for C57/BL6 strain) and human Jurkat T cell line. Cell viability levels after electroporation allowed further manipulations such as in vitro expansion and Chimeric Antigen Receptor (CAR) mediated gain of function for target cell lysis.
We describe here an efficient general protocol for electroporation based modification of T lymphocytes. By opening access to this protocol, we expect that efficient gene transfer to T lymphocytes, for transient or stable expression, may be achieved by an increased number of laboratories at lower and affordable costs.
基因转移到 T 淋巴细胞在历史上依赖于逆转录病毒和慢病毒，但最近基于转座子的基因转移作为一种更简单、更直接的方法来实现稳定的转基因表达而兴起。T 淋巴细胞的表达盒转移仍然具有挑战性，主要基于商业试剂盒。
我们在此报告一种基于自制缓冲液、通用比色皿和广泛使用的 Lonza 电穿孔仪 II 装置的便捷且经济实惠的方法，以促进 T 淋巴细胞的有效基因转移。
该方法在原代人 T 淋巴细胞（平均 45%，41-59%）、难转染的小鼠 T 细胞（C57/BL6 品系平均 38%，36-42%）和人 Jurkat T 细胞系中产生高水平的转基因表达。电穿孔后的细胞活力水平允许进一步进行操作，如体外扩增和嵌合抗原受体（CAR）介导的靶细胞裂解的功能获得。
我们在这里描述了一种用于 T 淋巴细胞电穿孔修饰的有效通用方案。通过开放对该方案的访问，我们期望更多的实验室能够以更低的成本实现高效的 T 淋巴细胞基因转移，无论是瞬时表达还是稳定表达。
