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In this article, we present several protocols that describe the steps from cloning and obtaining a large amount of pure plasmid DNA to generation of lentiviruses based on these constructs. The protocols have been worked out on human cell culture HEK293T but can be adapted for other cell cultures. This protocol was designed to be simple to execute and cheap since it requires only materials and consumables widely available in molecular laboratories, such as salts, alcohols, etc., and no complicated laboratory equipment. These protocols are highly effective and can be performed in any standard molecular biology laboratory.
Keywords: plasmid, mammalian cell transfection, lentiviral transduction, lentivirus, HEK293T, E. coli
Transfection is a procedure that introduces DNA and RNA into eukaryotic cells to produce genetically modified cells. Transfection is a powerful analytical tool for studying gene function and their regulation by enhancing or inhibiting specific gene expression in cells. It is also important for studying proteins or producing recombinant proteins in mammalian cells [1]. Transfection can be used in gene therapy, for production of recombinant proteins for therapeutic purposes, small interference RNA knockdown procedures [2] and is an essential step for lentivirus production [3].
There are various approaches for mammalian cell transfection. However, one of the key factors for successful transfection remains the quality and the quantity of the transfected material (DNA, RNA). To transfect human cells, a researcher needs a large amount of high-quality DNA. DNA midi- or maxiprep kits are often expensive and the amount of DNA isolated with their help is often insufficient.
In this study, we developed a new protocol suitable to achieve a high yield of plasmid DNA of high purity, acceptable for the transfection of mammalian cell cultures. Our method can also significantly reduce the cost of DNA isolation, since it provides a high yield of extracted plasmid without an implementation of commercial kits.
For this method, we used the E. coli bacterial strain STBL3 (ThermoFisher Scientific, Walthon, MA, USA). This strain was designed especially for cloning the direct repeats found in lentiviral expression vectors and it gives a higher yield of extracted DNA. However, the introduced protocol is probably compatible with any E. coli strain used for the cloning.
The DNA purity is sufficient for basic transfection methods using cationic polymers, such as PEI (polyethylenimine) or TurboFect (ThermoFisher, Walthon, MA, USA). Transfection using PEI has several advantages, like its cheapness and suitability for various cell cultures [4].
Lentiviral technology came to widespread use in molecular and cell biology for stable gene expression, gene silencing, generating transgene animals, induction of pluripotent stem cells, stem cell modification and lineage tracking, immunization and in vivo imaging [5]. Lentiviral technology is based on the co-transfection of human cell line HEK293T with packaging, envelope and transfer plasmids (containing the gene of interest). This is resulted in the assembly of the lentivirus, which, upon transduction, became a powerful tool for the expression of exogenous genes into various types of cells both in vitro and in vivo. Today, this method is the most effective way to obtain stable cell lines producing the studied genetic products [6].
These protocols can be easily reproduced in almost any lab, they have a good efficiency and may be necessary for the wide range of studies in cell and molecular biology.
In this article, we offer well-reproducible, high-performance protocols that are guided and adapted to work with mammalian cell cultures. This protocol of plasmid DNA extraction is also applicable to other techniques where a large amount of plasmid DNA is required.
In this article, we propose an optimized protocol for the isolation of plasmid DNA ( Figure 1 ) from bacterial cultures without the use of commercial kits for its subsequent application in genetic modifications of mammalian cell cultures. The advantages of this method are as follows: a high efficiency (concentration of plasmid DNA at the output is 3–8 mg/mL), availability, sufficient purity of the obtained material, as well as cheapness along with relatively low time costs. In addition, the protocol contains a description of the methods for efficient transfection and assembly of lentiviral particles based on the obtained plasmid DNA, which have been successfully tested for immortalized and primary mammalian cell cultures.
Experimental design. Scheme of (A) the experiment for the isolation of plasmid DNA; (B) the experiment for the mammalian cells transfection; (C) the experiment for the lentiviral vector production. All stages after the change of media on Day 4 should be carried out with the proper biosafety containment recommended for research with lentiviral vectors. Created with BioRender.com.
We recommend using plasmids containing fluorescent labels, as this will provide the best control over the experiment, calculation of transfection/transduction efficiency and viral assembly.
The plasmids obtained for the study were transformed into E. coli according to the Inoue standard protocol [7]. We used the plasmids pLenti-CMV-EGFP Hygro (656-4) [8], pMD2 (gift from Didier Trono (Addgene plasmid # 12259)) and PAX2 (gift from Didier Trono (Addgene plasmid # 12260)). The protocol is based on the Birnboim and Doly DNA extraction protocols with modifications [9,10].
This protocol lists the plasmids and strain used in this study, but the DNA extraction protocol is suitable for all strains and plasmids. It is suitable for all researchers working with cell cultures due to its simplicity and it does not require complicated equipment or expensive reagents.
