Generating green fluorescent mice by germline transmission of green fluorescent ES cells
Generating green fluorescent mice by germline transmission of green fluorescent ES cells
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DOI：
10.1016/s0925-4773(98)00093-8
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发表时间：
1998-08-01
影响因子：
2.6
通讯作者：
Nagy, A
中科院分区：
文献类型：
--
作者：
Hadjantonakis, AK;Gertsenstein, M;Nagy, A
绿色荧光蛋白（GFP）及其变体目前代表了唯一可用于通过转基因标记培养物中或多细胞生物体中的哺乳动物细胞的非侵入性标记物。迄今为止，该标记基因已被广泛用于许多生物的研究，但由于遇到弱荧光和野生型蛋白在较高温度下不稳定的问题，尚未发现在哺乳动物中的大规模应用。然而，最近，已经在野生型（wt）GFP中制备了几种突变体，以改善其热稳定性和荧光。EGFP（增强型GFP）是一种这样的wtGFP变体。作为评估EGFP在ES细胞介导的策略中的使用的第一步，我们已经建立了表达EGFP的小鼠胚胎干（ES）细胞系，其可以在培养中繁殖，重新引入小鼠中，或在体外诱导分化，同时仍然保持普遍的EGFP表达。结果表明：（1）在囊胚期预选嵌合体胚胎可能提高需要通过聚集嵌合体进行ES细胞生殖系传递的转基因方案的效率;（2）由在早期植入后发育期间有活性的启动子驱动的非侵入性标志物的表达，允许在胚胎发育的窗口期间接近胚胎，这在以前是难以研究的;（3）突变ES细胞的行为可以通过对包含绿色荧光细胞/组织的嵌合胚胎或成年动物的简单显微镜观察来跟踪;和（4）F1小鼠和随后几代动物的杂交表明，可以通过UV光对后代进行基因分型，使得转基因纯合的小鼠可以由于它们增加的荧光而与半合子区分开。(C)1998爱思唯尔科学爱尔兰有限公司保留所有权利。
Green fluorescent protein (GFP) and its variants currently represent the only non-invasive markers available for labeling mammalian cells in culture or in a multicellular organism through transgenesis. To date this marker gene has been widely used in the study of many organisms, but as yet has not found large-scale application in mammals due to problems encountered with weak fluorescence and instability of the wild-type protein at higher temperatures. Recently, though, several mutants have been made in the wild-type (wt) GFP so as to improve its thermostability and fluorescence. EGFP (enhanced GFP) is one such wtGFP variant. As a first step in assessing the use of EGFP in ES cell-mediated strategies, we have established a mouse embryonic stem (ES) cell lines expressing EGFP, which can be propagated in culture, reintroduced into mice, or induced to differentiate in vitro, while still maintaining ubiquitous EGFP expression. From the results presented we can suggest that: (1) possible improvements in the efficiency of transgenic regimes requiring the germline transmission of ES cells by aggregation chimeras can be made by the preselection chimeric embryos at the blastocyst stage; (2) the expression of a noninvasive marker, driven by a promoter that is active during early postimplantation development, allows access to embryos during a window of embryonic development that has previously been difficult to investigate; (3) the behavior of mutant ES cells can be followed with simple microscopic observation of chimeric embryos or adult animals comprising green fluorescent cells/tissues; and (4) intercrosses of Fl mice and subsequent generations of animals show that progeny can be genotyped by UV light, such that mice homozygous for the transgene can be distinguished from hemizygotes due to their increased fluorescence. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.