Kandarakov, O.; Polyakova, N.; Petrovskaya, A.; Bruter, A.; Belyavsky, A. CD52/FLAG and CD52/HA Fusion Proteins as Novel Magnetic Cell Selection Markers. Preprints2024, 2024050407. https://doi.org/10.20944/preprints202405.0407.v1
APA Style
Kandarakov, O., Polyakova, N., Petrovskaya, A., Bruter, A., & Belyavsky, A. (2024). CD52/FLAG and CD52/HA Fusion Proteins as Novel Magnetic Cell Selection Markers. Preprints. https://doi.org/10.20944/preprints202405.0407.v1
Chicago/Turabian Style
Kandarakov, O., Alexandra Bruter and Alexander Belyavsky. 2024 "CD52/FLAG and CD52/HA Fusion Proteins as Novel Magnetic Cell Selection Markers" Preprints. https://doi.org/10.20944/preprints202405.0407.v1
Abstract
At present, magnetic selection of genetically modified cells is mainly performed with surface markers naturally expressed by cells such as LNGFR, CD4 and 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 selection of CRISPR-modified cells using FACS. In the current study, we tested whether these markers can be used for 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 spurious co-isolation of negative cells.
Keywords
HA tag; FLAG tag; LNGFR; EGFP; DsRedExpress2; magnetic selection; MACS selection
Subject
Biology and Life Sciences, Cell and Developmental Biology
Copyright:
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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