Uckert W, Pedersen L, Günzburg W
Max-Delbrück-Center for Molecular Medicine, Berlin, Germany.
Methods Mol Med. 2000;35:275-85. doi: 10.1385/1-59259-086-1:275.
Genes-encoding marker proteins, which are easily assayable, are useful to monitor cell lineage, gene expression, or promoter activities. In gene-transfer technology such marker genes allow a direct and simple detection of successfully transduced cells. The detection of marker gene products such as β-galactosidase (β-gal), chloramphenicol acetyltransferase (CAT), alkaline phosphatase, or luciferase involves either cell fixation, which kills the cells or antibody-mediated detection, which is time consuming. Drug-resistance genes such as neomycin, puromycin, hygromycin, or zeocin allow a positive selection of transduced cells, but require days to weeks of growth in selective media. Moreover, these genes can change the growth characteristics of the transduced cells through terminal differentiation or can interfere with the expression of the gene of interest (1). Therefore, a marker gene system that provides timely, accurate, and nontoxic detection of successfully transduced living cells would be of great advantage. One interesting candidate gene that fulfills these requirements is the gene-encoding green fluorescent protein (GFP). It was originally isolated from the jellyfish Aquorea victoria. The GFP cDNA consists of 730 bp, which encode a 238 amino acid protein with a molecular weight of 27 kD (2). Wild-type GFP emits a vibrant green fluorescence upon exposure to blue light (450-490 nm). The signal is detectable by fluorescence microscopy and fluorescence-activated cell sorting (FACS) (3). Because the fluorescence of wild-type GFP after excitation is not strong enough for many applications, different variants of GFP have been developed. In one such variant, a point mutation was introduced at amino acid 65 (GFP-S65T) leading to a "red-shifted" excitation maximum with an approximately five-fold stronger fluorescent intensity (4). In a further variant, the "red-shifted" GFP was "humanized" by the introduction of numerous silent mutations that alter the codons to those more commonly used in human genes resulting in the improved translation of the gene (5-7). An additional point mutation at amino acid 64 in which phenylalanine was altered to leucine (F64L) further enhances gene expression (8). GFP has been expressed without cytotoxic effects in different organisms and is of special interest as a marker for monitoring cell lines and gene expression (3). The application of GFP in gene-transfer protocols allows the simple detection of transduced cells and offers the possibility for immediate enrichment of viable transduced cells by FACS (3,9,10). This is of great interest in gene transfer into poorly transducable cells, e.g., hematopoietic stem and progenitor cells.
编码易于检测的标记蛋白的基因，对于监测细胞谱系、基因表达或启动子活性很有用。在基因转移技术中，此类标记基因能直接且简单地检测成功转导的细胞。对标记基因产物如β-半乳糖苷酶（β-gal）、氯霉素乙酰转移酶（CAT）、碱性磷酸酶或荧光素酶的检测，要么涉及杀死细胞的细胞固定，要么涉及耗时的抗体介导检测。新霉素、嘌呤霉素、潮霉素或博来霉素等耐药基因能对转导细胞进行阳性选择，但在选择性培养基中需要数天到数周的生长时间。此外，这些基因可通过终末分化改变转导细胞的生长特性，或干扰目的基因的表达（1）。因此，一个能对成功转导的活细胞进行及时、准确且无毒检测的标记基因系统将具有很大优势。满足这些要求的一个有趣候选基因是编码绿色荧光蛋白（GFP）的基因。它最初是从维多利亚多管水母中分离出来的。GFP cDNA由730个碱基对组成，编码一个分子量为27 kD的238个氨基酸的蛋白质（2）。野生型GFP在蓝光（450 - 490 nm）照射下发出明亮的绿色荧光。该信号可通过荧光显微镜和荧光激活细胞分选（FACS）检测到（3）。由于野生型GFP激发后的荧光强度对许多应用来说不够强，因此已开发出不同的GFP变体。在一种这样的变体中，在第65位氨基酸处引入了一个点突变（GFP - S65T），导致激发最大值出现“红移”，荧光强度增强约五倍（4）。在另一种变体中，通过引入大量沉默突变将“红移”GFP进行了“人源化”，这些突变将密码子改变为人类基因中更常用的密码子，从而提高了基因的翻译效率（5 - 7）。在第64位氨基酸处的另一个点突变，将苯丙氨酸改变为亮氨酸（F64L），进一步增强了基因表达（8）。GFP已在不同生物体中无细胞毒性地表达，作为监测细胞系和基因表达的标记物特别受关注（3）。GFP在基因转移方案中的应用允许简单地检测转导细胞，并提供了通过FACS立即富集存活转导细胞的可能性（3,9,10）。这对于将基因转移到难以转导的细胞，如造血干细胞和祖细胞中非常有意义。
