Cassettes for PCR-mediated construction of green, yellow, and cyan fluorescent protein fusions in Candida albicans
Cassettes for PCR-mediated construction of green, yellow, and cyan fluorescent protein fusions in Candida albicans
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作者：
Gerami-Nejad, M;Berman, J;Gale, CA
我们已经开发了一套含有荧光蛋白盒的质粒，用于PCR介导的白念珠菌基因标记。我们设计了针对C.白色念珠菌密码子使用。与C.白色念珠菌营养缺陷型标记序列，通过PCR扩增并直接转化到酵母中。将基因特异性序列掺入PCR引物中，使得标签盒通过同源重组整合在感兴趣的基因的3 '端。该技术用于用GFP、YFP和CFP标记Cdc3和Tub1，其容易通过荧光显微镜观察到并且如预期定位。此外，Tub1-YFP和Cdc3-CFP在相同的细胞中观察到。因此，该技术指导了多荧光蛋白融合体的一步构建，促进了蛋白在C.白念珠菌细胞。版权所有（C）2001约翰威利父子有限公司
We have developed a set of plasmids containing fluorescent protein cassettes for use in PCR-mediated gene tagging in Candida albicans. We engineered YFP and CFP variants of the CFP sequence optimized for C. albicans codon usage. The fluorescent protein sequences, linked to C. albicans auxotrophic marker sequences, Ir ere amplified by PCR and transformed directly into yeast. Gene-specific sequence was incorporated into the PCR primers, such that the tag-cassette integrates by homologous recombination at the 3'-end of the gene of interest, This technique was used to tag Cdc3 and Tub1 with GFP, YFP and CFP, which were readily visualized by fluorescence microscopy and localized as expected, In addition, Tub1-YFP and Cdc3-CFP were visualized in the same cells. Thus, this technique directs one-step construction of multiple fluorescent protein fusions, facilitating the study of protein co-expression and co-localization in C. albicans cells in vivo. Copyright (C) 2001 John Wiley & Sons, Ltd.