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The Prodigious Potential of mRNA Electrotransfer as a Substitute to Conventional DNA-Based Transient Transfection

Théo Juncker

Bruno Chatton

and

Mariel Donzeau

Cells 2023, 12(12), 1591; https://doi.org/10.3390/cells12121591 - 08 Jun 2023

Cited by 1 | Viewed by 739

Abstract

Transient transfection of foreign DNA is the most widely used laboratory technique to study gene function and product. However, the transfection efficiency depends on many parameters, including DNA quantity and quality, transfection methods and target cell lines. Here, we describe the considerable advantage of mRNA electroporation compared to conventional DNA-based systems. Indeed, our methodology offers extremely high transfection efficiency up to 98% regardless of the cell line tested. Protein expression takes place a few hours post-transfection and lasts over 72 h, but overall, the electrotransfer of mRNAs enables the monitoring of the level of protein expressed by simply modulating the amount of mRNAs used. As a result, we successfully conducted cell imaging by matching the levels of expressed V_HHs and the antigen present in the cell, preventing the necessity to remove the excess unbound V_HHs. Altogether, our results demonstrate that mRNA electrotransfer could easily supplant the conventional DNA-based transient expression system. Full article

(This article belongs to the Special Issue Advances in Cell Techniques)

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Open AccessArticle

Selection of Cell Populations with High or Low Surface Marker Expression Using Magnetic Sorting

Natalia Polyakova

Oleg Kandarakov

and

Alexander Belyavsky

Cells 2023, 12(9), 1286; https://doi.org/10.3390/cells12091286 - 29 Apr 2023

Viewed by 998

Abstract

Magnetic cell sorting technology stands out because of its speed, simplicity, and ability to process large cell numbers. However, it also suffers from a number of drawbacks, in particular low discrimination power, which results in all-or-none selection outcomes limited to a bulk separation of cell populations into positive and negative fractions, as well as the modest purity of the selected cells and the inability to select subpopulations of cells with high expression of a surface marker. In the present study, we developed a simple solution to this problem and confirmed the effectiveness of this approach by multiple experiments with the magnetic selection of transduced cell populations. Murine NIH 3T3 cells were transduced with the bicistronic retroviral vector constructs co-expressing fluorescent reporter proteins EGFP (enhanced green fluorescent protein) or DsRed-Express 2 and LNGFR (low-affinity nerve growth factor receptor) as surface selection markers. The effects of the magnetic selection of transduced cells with anti-LNGFR Micro Bead (MB) doses ranging from 0.5 to 80 µL have been assessed. Low doses of MBs favored the depletion of weakly positive cells from the population, resulting in the higher expression levels of EGFP or DsRed-Express2 reporters in the selected cell fractions. Low MB doses also contributed to the increased purity of the selected population, even for samples with a low initial percentage of positive cells. At the same time, high MB doses resulted in the increased yield and a more faithful representation of the original expression profiles following selection. We further demonstrate that for populations with fairly narrow distribution of expression levels, it is possible to achieve separation into high- and low-expressing subsets using the two-stage selection scheme based on the sequential use of low and high doses of MBs. For populations with broad expression distribution, a one-stage selection with low or high doses of MBs is sufficient for a clear separation of low- and high-expressing subsets in the column-retained and flow-through fractions, respectively. This study substantially extends the potential of magnetic cell sorting, and may open new possibilities in a number of biomedical applications. Full article

(This article belongs to the Special Issue Advances in Cell Techniques)

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Open AccessArticle

Systematic Methods for Isolating High Purity Nuclei from Ten Important Plants for Omics Interrogation

and

Cells 2022, 11(23), 3919; https://doi.org/10.3390/cells11233919 - 03 Dec 2022

Cited by 3 | Viewed by 2231

Abstract

Recent advances in developmental biology have been made possible by using multi-omic studies at single cell resolution. However, progress in plants has been slowed, owing to the tremendous difficulty in protoplast isolation from most plant tissues and/or oversize protoplasts during flow cytometry purification. Surprisingly, rapid innovations in nucleus research have shed light on plant studies in single cell resolution, which necessitates high quality and efficient nucleus isolation. Herein, we present efficient nuclei isolation protocols from the leaves of ten important plants including Arabidopsis, rice, maize, tomato, soybean, banana, grape, citrus, apple, and litchi. We provide a detailed procedure for nucleus isolation, flow cytometry purification, and absolute nucleus number quantification. The nucleus isolation buffer formula of the ten plants tested was optimized, and the results indicated a high nuclei yield. Microscope observations revealed high purity after flow cytometry sorting, and the DNA and RNA quality extract from isolated nuclei were monitored by using the nuclei in cell division cycle and single nucleus RNA sequencing (snRNA-seq) studies, with detailed procedures provided. The findings indicated that nucleus yield and quality meet the requirements of snRNA-seq, cell division cycle, and likely other omic studies. The protocol outlined here makes it feasible to perform plant omic studies at single cell resolution. Full article

(This article belongs to the Special Issue Advances in Cell Techniques)

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