Maloshenok Liliya, Abushinova Gerel, Kazachkina Natalia, Bogdanov Alexei, Zherdeva Victoria
Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, 33, Bldg. 2 Leninsky Ave., 119071 Moscow, Russia.
Vavilov Institute of General Genetics of the Russian Academy of Sciences, 117971 Moscow, Russia.
Materials (Basel). 2023 Jan 19;16(3):940. doi: 10.3390/ma16030940.
The catalytically inactive mutant of Cas9 (dCas9) endonuclease has multiple biomedical applications, with the most useful being the activation/repression of transcription. dCas9 family members are also emerging as potential experimental tools for gene mapping at the level of individual live cells and intact tissue. We performed initial testing on a set of tools for Cas9-mediated visualization of nuclear compartments. We investigated doxycycline (Dox)-inducible (Tet-On) intracellular distribution of constructs encoding dCas9 orthologs from St. thermophilus (St) and N. meningitides (Nm) fused with EGFP and mCherry fluorescent proteins (FP) in human A549 cells. We also studied time-dependent expression of these chimeric fluorescent constructs (dCas9-FP) after Tet-On induction in live cells and compared it with the time course of dCas9-FP expression in experimental dCas9-FP-expressing tumor xenografts using a combination of fluorescence imaging and in vivo contrast-assisted magnetic resonance imaging for assessing the extent of tumor perfusion. In vivo Dox-induction of mCherry-chimera expression occurred in tumor xenografts as early as 24 h post-induction and was visualized by using optical clearing (OC) of the skin. OC via topical application of gadobutrol enabled high-contrast imaging of FP expression in tumor xenografts due to a 1.1-1.2-fold increase in FI in both the red and green channels.
Cas9核酸内切酶的催化失活突变体（dCas9）具有多种生物医学应用，其中最有用的是转录激活/抑制。dCas9家族成员也正成为在单个活细胞和完整组织水平进行基因定位的潜在实验工具。我们对一组用于Cas9介导的核区室可视化的工具进行了初步测试。我们研究了强力霉素（Dox）诱导型（Tet-On）在人A549细胞中编码与EGFP和mCherry荧光蛋白（FP）融合的嗜热栖热菌（St）和脑膜炎奈瑟菌（Nm）的dCas9直系同源物的构建体的细胞内分布。我们还研究了这些嵌合荧光构建体（dCas9-FP）在Tet-On诱导后在活细胞中的时间依赖性表达，并使用荧光成像和体内对比辅助磁共振成像相结合的方法，将其与实验性表达dCas9-FP的肿瘤异种移植中dCas9-FP表达的时间进程进行比较，以评估肿瘤灌注程度。在肿瘤异种移植中，早在诱导后24小时就发生了体内Dox诱导的mCherry嵌合体表达，并通过皮肤光学清除（OC）进行可视化。通过局部应用钆布醇进行OC，由于红色和绿色通道中的荧光强度增加了1.1-1.2倍，使得能够对肿瘤异种移植中的FP表达进行高对比度成像。
