Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov Str. 32, 119991 Moscow, Russia.
Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Vavilov Str. 32, 119991 Moscow, Russia.
Int J Mol Sci. 2024 Jun 8;25(12):6353. doi: 10.3390/ijms25126353.
At present, the magnetic selection of genetically modified cells is mainly performed with surface markers naturally expressed by cells such as CD4, LNGFR (low affinity nerve growth factor receptor), and MHC class I molecule H-2Kk. The disadvantage of such markers is the possibility of their undesired and poorly predictable expression by unmodified cells before or after cell manipulation, which makes it essential to develop new surface markers that would not have such a drawback. Earlier, modified CD52 surface protein variants with embedded HA and FLAG epitope tags (CD52/FLAG and CD52/HA) were developed by the group of Dr. Mazurov for the fluorescent cell sorting of CRISPR-modified cells. In the current study, we tested whether these markers can be used for the magnetic selection of transduced cells. For this purpose, appropriate constructs were created in MigR1-based bicistronic retroviral vectors containing EGFP and DsRedExpress2 as fluorescent reporters. Cytometric analysis of the transduced NIH 3T3 cell populations after magnetic selection evaluated the efficiency of isolation and purity of the obtained populations, as well as the change in the median fluorescence intensity (MFI). The results of this study demonstrate that the surface markers CD52/FLAG and CD52/HA can be effectively used for magnetic cell selection, and their efficiencies are comparable to that of the commonly used LNGFR marker. At the same time, the significant advantage of these markers is the absence of HA and FLAG epitope sequences in cellular proteins, which rules out the spurious co-isolation of negative cells.
目前，基因修饰细胞的磁选主要通过细胞表面标志物进行，如 CD4、LNGFR（低亲和力神经生长因子受体）和 MHC Ⅰ类分子 H-2Kk。这些标志物的缺点是，在细胞操作之前或之后，未修饰的细胞可能会不期望地、难以预测地表达这些标志物，这使得开发新的表面标志物变得非常必要，而这些新标志物则不会有这样的缺点。早些时候，Mazurov 博士的研究小组开发了具有嵌入式 HA 和 FLAG 表位标签的修饰 CD52 表面蛋白变体（CD52/FLAG 和 CD52/HA），用于 CRISPR 修饰细胞的荧光细胞分选。在本研究中，我们测试了这些标记物是否可用于转导细胞的磁选。为此，在基于 MigR1 的双顺反子逆转录病毒载体中创建了适当的构建体，其中包含 EGFP 和 DsRedExpress2 作为荧光报告基因。磁选后转导 NIH 3T3 细胞群体的细胞仪分析评估了分离效率和获得群体的纯度，以及中荧光强度（MFI）的变化。这项研究的结果表明，表面标记物 CD52/FLAG 和 CD52/HA 可有效地用于磁选细胞，其效率可与常用的 LNGFR 标记物相媲美。同时，这些标记物的一个显著优势是细胞蛋白中不存在 HA 和 FLAG 表位序列，从而排除了阴性细胞的意外共分离。
