Ramiro A R, De Yébenes V G, Trigueros C, Carrasco Y R, Toribio M L
Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid, Cantoblanco, Spain.
Hum Gene Ther. 1998 May 1;9(7):1103-9. doi: 10.1089/hum.1998.9.7-1103.
Owing to its autofluorescence properties, green fluorescent protein (GFP) has aroused increasing interest as a marker system for many research applications. In this study we investigated the suitability of the "enhanced" GFP (EGFP), a mutant version of GFP optimized for flow cytometry and microscopy detection, as a reporter gene for retroviral transduction protocols. EGFP was shown to display a bright and stably maintained emission pattern in transfected GP+envAm12 packaging cells. Stable fluorescent emission was observed as well after transduction in NIH 3T3 fibroblasts and in the human Jurkat T cell line, in which EGFP was shown to confer no deleterious effect or growth disadvantage on the expressing cells. Moreover, EGFP expression could be detected after short-term retroviral exposure, thus allowing a rapid and quantitative retroviral titering assay, alternative to the standard colony-formation procedure. Most importantly, we showed the feasibility of EGFP as a marker gene in retroviral-mediated transduction of primary lymphoid precursors. In particular, transduction of CD34+CD1- human thymocytes by short-term cocultivation yielded up to 30% of EGFP-expressing cells, while maintaining CD34 expression levels. Finally, when cultured under multicytokine-supported conditions, such transduced intrathymic progenitors were shown to efficiently generate lymphoid-related dendritic cells, which displayed a distinct EGFP expression. Therefore, because of its rapid and easy detectability and its nontoxic characteristics, EGFP proves itself to be a valuable reporter gene by allowing the transduction of multipotential progenitors and by being compatible with the developmental programs of lymphoid lineage generation.
由于其自身荧光特性，绿色荧光蛋白（GFP）作为一种标记系统在许多研究应用中引起了越来越多的关注。在本研究中，我们调查了“增强型”GFP（EGFP）作为逆转录病毒转导方案的报告基因的适用性，EGFP是GFP的一种突变体，针对流式细胞术和显微镜检测进行了优化。EGFP在转染的GP+envAm12包装细胞中显示出明亮且稳定维持的发射模式。在NIH 3T3成纤维细胞和人Jurkat T细胞系中转导后也观察到稳定的荧光发射，其中EGFP对表达细胞没有有害影响或生长劣势。此外，在短期逆转录病毒暴露后可以检测到EGFP表达，从而允许进行快速定量的逆转录病毒滴度测定，这是标准集落形成程序的替代方法。最重要的是，我们展示了EGFP作为标记基因在原代淋巴前体细胞逆转录病毒介导的转导中的可行性。特别是，通过短期共培养对CD34+CD1-人胸腺细胞进行转导，产生了高达30%的表达EGFP的细胞，同时保持了CD34表达水平。最后，当在多种细胞因子支持的条件下培养时，这种转导的胸腺内祖细胞被证明能够有效地产生淋巴相关的树突状细胞，这些细胞显示出明显的EGFP表达。因此，由于其快速且易于检测以及无毒的特性，EGFP通过允许多能祖细胞的转导并与淋巴谱系生成的发育程序兼容，证明了自己是一种有价值的报告基因。
