PDF(Pubmed)
Abstract：
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.
摘要：
目前,遗传修饰细胞的磁性选择主要是通过细胞天然表达的表面标记进行的，例如CD4，LNGFR（低亲和力神经生长因子受体），和MHCI类分子H-2Kk。这些标记的缺点是在细胞操作之前或之后,它们可能被未修饰的细胞不期望的和难以预测的表达。这使得必须开发新的表面标记，不会有这样的缺点。早些时候，Mazurov博士小组开发了具有嵌入HA和FLAG表位标签的修饰的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.在目前的研究中,我们测试了这些标记是否可以用于转导细胞的磁性选择。为此,在含有EGFP和DsRedExpress2作为荧光报道分子的基于MigR1的双顺反子逆转录病毒载体中创建适当的构建体.磁性选择后转导的NIH3T3细胞群的细胞计数分析评估了分离效率和获得的群体的纯度，以及中值荧光强度（MFI）的变化。这项研究的结果表明，表面标志物CD52/FLAG和CD52/HA可以有效地用于磁性细胞选择，它们的效率与常用的LNGFR标记相当。同时,这些标记的显著优势是细胞蛋白中缺乏HA和FLAG表位序列，这排除了阴性细胞的假的共同隔离。
