Mizutani Akiko, Ohtsuka Masato, Kimura Minoru, Tanaka Masafumi, Inoko Hidetoshi
Depertment of Molecular Life Sciences, Division of Basic Molecular Science and Molecular Medicine, Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa 259-1193, Japan.
Nucleic Acids Symp Ser (Oxf). 2005(49):297-8. doi: 10.1093/nass/49.1.297.
The Cre-loxP system has been used for genetic engineering in many organisms ranging from E. coli to mouse. It is known that intermolecular recombination between two loxP sites is substantially less frequent than intramolecular recombination between the same sites. Therefore, for the efficient isolation of intermolecular loxP recombination products, it is essential to develop a powerful screening method. Here, we describe the construction of an EYFP variant (EYFP157) that could become a vital tool in such a screening method. EYFP157 contains, between Gln157 and Lys158, the JT15:JTZ17 mutant loxP sequence that is not cleaved by the Cre protein. When EYFP157 was expressed at 37 degrees C in E. coli and mouse cells, fluorescence of the protein was readily detectable under a standard microscope. EYFP157 possessed 1-3% of the fluorescence from the wild-type EYFP. Interestingly, at room temperature EYFP157 fluoresced more efficiently in E. coli cells and thus the difference in fluorescence intensity between EYFP157 and the wild-type EYFP became negligible. The potential application of EYFP157 as a recombination indicator in both E. coli and mouse cells is further discussed.
Cre-loxP系统已被用于从大肠杆菌到小鼠等多种生物体的基因工程。已知两个loxP位点之间的分子间重组比同一位点之间的分子内重组频率低得多。因此，为了有效地分离分子间loxP重组产物，开发一种强大的筛选方法至关重要。在此，我们描述了一种增强型黄色荧光蛋白变体（EYFP157）的构建，它可能成为这种筛选方法中的重要工具。EYFP157在Gln157和Lys158之间包含未被Cre蛋白切割的JT15:JTZ17突变loxP序列。当EYFP157在大肠杆菌和小鼠细胞中于37℃表达时，在标准显微镜下很容易检测到该蛋白的荧光。EYFP157具有野生型EYFP荧光的1%-3%。有趣的是，在室温下，EYFP157在大肠杆菌细胞中荧光效率更高，因此EYFP157与野生型EYFP之间的荧光强度差异变得可以忽略不计。进一步讨论了EYFP157作为大肠杆菌和小鼠细胞中重组指示剂的潜在应用。
