Limón A, Briones J, Puig T, Carmona M, Fornas O, Cancelas J A, Nadal M, García J, Rueda F, Barquinero J
Department of Cryobiology, Institut de Recerca Oncològica, Barcelona, Spain.
Blood. 1997 Nov 1;90(9):3316-21.
Retroviral vectors constitute the most efficient system to deliver and integrate foreign genes into mammalian cells. We have developed a producer cell line that yields high titers of amphotropic retroviral vectors carrying the enhanced green fluorescent protein (EGFP) gene, a codon humanized, red-shifted variant of the green fluorescent protein (GFP) gene, which can be used as a selectable marker. We have used a hybrid vector that has been shown to efficiently drive gene expression in hematopoietic cells. Virtually all murine and human cell lines and primary human hematopoietic cells tested were transduced with varying efficiency after incubation with vector-containing supernatants. Human CD34(+) cells obtained from cord blood or aphereses products were transduced using a protocol that involves daily addition of vector-containing supernatants for 6 consecutive days. At day 6, up to 16% of the cells expressed EGFP, as assessed by flow cytometry. Sorted EGFP-expressing cells were able to produce fluorescent hematopoietic colonies. EGFP's main advantages are its fast flow cytometry determination and the possibility of cell sorting and simultaneous evaluation of the transduction efficiency along with other phenotypic markers.
逆转录病毒载体是将外源基因导入哺乳动物细胞并实现整合的最有效系统。我们开发了一种生产细胞系，该细胞系可产生高滴度的携带增强型绿色荧光蛋白（EGFP）基因的双嗜性逆转录病毒载体，EGFP是绿色荧光蛋白（GFP）基因的密码子人源化、红移变体，可作为选择标记。我们使用了一种已被证明能有效驱动造血细胞中基因表达的杂交载体。几乎所有测试的小鼠和人类细胞系以及原代人类造血细胞在与含载体的上清液孵育后都能以不同效率被转导。从脐血或单采血液成分制品中获得的人类CD34(+)细胞采用一种方案进行转导，该方案包括连续6天每天添加含载体的上清液。在第6天，通过流式细胞术评估，高达16%的细胞表达EGFP。分选的表达EGFP的细胞能够产生荧光造血集落。EGFP的主要优点是其通过流式细胞术测定速度快，并且有可能进行细胞分选以及与其他表型标记同时评估转导效率。
