Having trouble with cell transfection? Did you pick the right method?

Having trouble with cell transfection? Did you pick the right method?

Transfection is a very common operation in cell experiments. Through transfection, foreign nucleic acids (DNA, RNA, etc.) can be introduced into eukaryotic cells to study the function of genes, which is an essential step in the construction of gene-editing cell lines. However, the problem of low transfection efficiency always bothers many novices, and the transfection of a new cell line is usually challenging for an experienced scientist.

What factors are involved in the efficiency of cell transfection? Generally speaking, it can be summarized into three aspects:

1. Cells: Including the type, source and passage of the cell line, which basically determines whether the cell line is easy to be transfected.

2. Transfection vector: The size, quality and quantity of the transfection vector also have impacts on the transfection efficiency. For vectors of different sizes of the vectors, the experimental parameters are usage should be optimized. In terms of quality, we should control the concentration, purity and conformation of the plasmid, and check whether RNA is degraded.

3. Transfection methods and reagents: no one transfection method is applicable to all cell lines. For different cell lines, it is important to select appropriate transfection methods and reagents.

In this article, we will summarize the transfection methods and reagents, and share our experience with you.

First of all, let's take a quick look at the methods of cell transfection. According to the principle of transfection, it can be divided into three categories: physical mediation (such as electroporation, microinjection, and particle bombardment), chemical mediation (such as calcium phosphate co-precipitation, liposome transfection, and cationic polymer mediation), and biological mediation (mainly various viruses transduction). Among them, the most commonly used methods in laboratories are electroporation, liposome transfection and lentivirus method.

Liposome transfection:

Liposomes form a complex containing DNA molecules through the interaction between the positive charges on the surface and the negative charges of nucleic acid phosphate groups. The complex is positively charged on the surface and will be adsorbed by the negatively charged cell membrane, and then complete the introduction of foreign DNA molecules through fusion or cell endocytosis, which is one of the most convenient transfection methods in the laboratory at present.

Following is the detail process:

1) When the transfection reagent and nucleic acid are incubated in vitro, a positively charged complex is formed.

2) The complex is added to the cells and binds to the negatively charged cell surface through electrostatic interaction.

3) The cells internalize the complex into the membrane vesicles of the endosome through endocytosis.

4) Transfection reagent made the endosome membrane unstable.

5) The complex escapes from the endosome and releases nucleic acids in the cytoplasm (siRNA, miRNA and large RNA are usually active in the cytoplasm).

6) DNA must be located in the nucleus to express and transcript.

Figure 1. Liposome transfection process ^[1]

Electroporation:

The electroporation causes the change of cell membrane potential through high intensity electric field, instantly improves the permeability of cell membrane, and makes the cell membrane produce reversible pores to facilitate the entry of foreign nucleic acids (Figure 2). It is applicable to almost all types of cells. At present, the commonly used electroporation systems on the market include Neon of Thermo, Nucleofector series of Lonza, Bio-rad Gene Pulser Xcell system, and Celetrix.

Figure 2. Principle of electroporation ^[2]

Lentivirus mediated transduction:

Lentivirus can transduce non-dividing cells and dividing cells. Human immunodeficiency virus type 1 (HIV-1) is the most studied lentivirus. The specific process is as follows:

1) The target plasmid of genetic engineering transformation and the helping plasmids needed for virus packaging are transferred to 293T cells to produce lentivirus particles needed for cell transduction.

2) Lentivirus particles infect target cells, and lentivirus surface envelope protein combined with cell receptors to get into cells.

3) Lentivirus RNA is reverse transcribed into double-stranded DNA.

4) Double-stranded DNA and integrase form a pre-integration complex, and then enter the nucleus through the nuclear pore.

5) Integrase integrates LTR-flanked sequence into cell genome.

6) The integrated foreign DNA starts to transcribe and express the target gene.

Figure 3. Lentivirus production and transduction process ^[3]

After knowing the principles of the three transfection methods, here we summarized their advantages and disadvantages.

So how can you find out which transfection method is best for your experiment?

1. Understanding the cell line: First, we need to sort out the cell background, the source, cell characteristics, culture conditions, current passage, subculture period and ratio, etc.

2. Reference transfection cases: finding successful transfection cases for reference can help you avoid detours! On the one hand, you can communicate with the transfection reagent supplier or electroporation instrument supplier to ask the efficiency and parameters for a certain cell line. On the other hand, you can search for the literature to check for the methods used in this cell line, and the detailed process. In addition, it is preferred to choose the transfection method according to the existing instruments and reagents of the laboratory, and perform efficiency tests.

3. Preliminary tests: With the above preparations, we can start the preliminary experiments to find out the best transfection parameters. Generally, the carrier with fluorescent label is used to test the transfection rate. Carry out transfection test on cells according to the data you get from the above step or the transfection protocol provided by the supplier. During the test, generally measure multiple groups of experimental parameters (such as different ratios of DNA and transfection reagents by liposome method, different parameters by electroporation, and different MOI by lentivirus method). Observe the transfection rate 24h to 48h after transfection, and select the group with the highest efficiency and good cell status for subsequent formal experiments. If the transfection efficiency of the highest group is less than 30%, it indicates that this method is not applicable to this cell line, and it is recommended to replace other transfection methods.

After reading the above steps, do you want to try it yourself? Ubigene provides in-stock lentivirus and transfection medium to help you test the transfection efficiency!

If you think it is time-consuming and laborious to explore the transfection parameters in the preliminary tests, you can also choose to hand over the tedious and repetitive cell construction work to Ubigene. We have rich experience in cell engineering and gene editing!

Ubigene is sophisticated in all kinds of transfection methods. Not only good at the methods mentioned above, but also master other transfection methods such as adenovirus (ADV), adeno-associated virus (AAV), and has established a database of more than 200 commonly used cell lines, including the data of transfection parameters, single-cell clone formation, and antibiotic screening. For the cell line we did not work with, we have a mature procedure to find out the best method to generate the customized cell line.

With the help of the transfection culture medium, we can achieve efficient transfection in some difficult-to-transfect cells, such as THP-1, RAW264.7, MDA MB-231, iPS/ES, and lay a good foundation for the follow-up work of stable cell line construction!

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Reference:

[1] https://www.mirusbio.com/transfection

[2] Du X, Wang J, Zhou Q, Zhang L, Wang S, Zhang Z, Yao C. Advanced physical techniques for gene delivery based on membrane perforation. Drug Deliv. 2018 Nov;25(1):1516-1525. doi: 10.1080/10717544.2018.1480674. PMID: 29968512; PMCID: PMC6058615.

[3] Maes M E, Colombo G, Schulz R, et al. Targeting microglia with lentivirus and AAV: Recent advances and remaining challenges[J]. Neuroscience letters, 2019, 707: 134310.