“Under the influence of the electrical current, temporary pores created into the cell membrane enables the entry of nucleic acid in a cell, the process is referred to as electroporation.”
Apart from the nucleic acid, the present method is powerful enough to transfer chemicals and drugs into cells. Therefore, the electroporation technique is also known as electrotransfer.
Electropermiabilization is another name used to describe the present method, let me tell you why!
Electrical pulse performs two functions here, permeabilize cells and creates pores in the cell membrane as well. Both functions help in gene transfer.
Gene therapy is nowadays very popular for curing inherited genetic disorders. Broadly, a nucleic acid sequence of interest is inserted into the target genome using either viral or non-viral vectors.
The gene replaces the mutant gene in a target cell. In the final stage, the transformed cells are cultured in vitro.
What usually we require to transfer a gene into a cell?
A target gene/ DNA or nucleic acid, vector, target cell and a technique to insert nucleic acid into a cell.
Our major focus here is on applications of electroporation in genetics. Hence we will discuss the process of how foreign DNA or plasmid DNA can be transferred into cells using the electroporation.
Further, we will also talk on principle, it’s applications and limitations.
Key Topics:
What is electroporation?
The term “electrophoration” was first coined by Eumann and co-workers in 1982. They had used the electrical current to break the cell membrane. From their discovery, the era of using the electrical pulse in the biotechnology was started.
Definition:
“Using the electrical current any biological molecules such as nucleic acid, drug, chemical or viral DNA can be inserted into a live cell by creating temporary pores in the cell membrane. The technique is known as electroporation or electropermeabilization.”
It’s an incredible biotechnology tool besides, PCR, DNA sequencing and vectors.
In genetics or molecular genetics, the present method is mostly used for transferring a gene, DNA sequence, unknown nucleic acid, viral DNA or plasmid DNA in any cell.
Thus, electroporation is employed for in vitro transfection experiments.
Any cell types like mammalian cells, live cells, bacterial cells or any other cells can be effectively used in it.
Principle:
The eukaryotic cell membrane or cell wall is made up of lipids, phospholipids, proteins and chitin or pectin, mostly.
Notably, the pectin or chitin is present in plant cells only. Furthermore, animal cells don’t have a cell wall. Instead of that, a smooth cell membrane protects the cytoplasm of animal cells.
Once we apply current to the cell suspension it creates some pores in the cell membrane.
1.0-1.5kV current can apply for effective insertion only for some milliseconds. Now, the cells become competent.
The competent cells are now ready for the intake of foreign material. Here, for instance, a DNA.
The nucleic acid-DNA or plasmid, transferred into the cell cytoplasm with the current. Immediately, after DNA insertion, cell pores closed by switching off the current.
The success rate of the entire process depends on two variables; pulse length and field strength.
This is an entire overview of electroporation for any application.
Process:
We will discuss the entire process of the present method using the example of gene transfer. It is also known as pulse generator too.
In the very first step, the plasmid is constructed for transformation. A gene of interest along with the marker DNA sequence ligated into the plasmid.
The selection of plasmid depends on the type and the size of nucleic acid used for gene therapy.
A population of target cells is taken into the suspension and filled with the electroporation buffer. The electroporation buffer protects cell suspension from mechanical damage.
Along with it, target DNA or plasmid DNA taken into the cuvette. Then it is placed in an electroporator.
Electroporation machine/ device:
As we said, the electroporation machine or device is known as electroporator. On either side of the cuvette, two aluminium electrodes are fitted.
The device is connected with the power source, when it switches on, the circuit completes.
Electrical pulses pass through the cell suspension. This will create temporary pores in the cell membrane.
Notably, the entire process completes within a second (in a microsecond or millisecond).
Charged molecules like DNA can pass through pores into the cytoplasm of the cell, under the influence of current.
After the completion of the entire process, the viability of cells is checked. In the final step, the transformed cells are cultured.
For in vitro cell culture, ready to use culture media can be used.
Now using DNA sequencing or Real-time PCR assay, results of experiments are determined. Also, the expression of the inserted gene can be determined.
Once, validation is done, now the cells are ready for injection. Using the present method, Gene knockout and knockout mice construction performed.
Notably, here apart from electroporation, viral- vectors like AAV, retrovirus or lentivirus can be used.
Further, non-viral vectors like liposomes, chemicals and other apoplexy methods can also be used.
But, the present method is one of the first choices for gene transfer and gene therapy experiments because of its tremendous advantages.
Related: What is Gene Therapy? and How Does it Work?
Advantages:
One of the major advantages of the present method is its high effectiveness and rapid transfection rate.
Electroporation can transfer foreign material effectively, faster than any other viral or non-viral vector-mediated delivery system.
In comparison with the viral vectors, It’s safer. The chance of infection is negligible.
Also, it’s non-toxic, non-allergic and non-mutagenic.
Transfection or transformation can perform on any living cells like mammalian, plant, bacteria, algae and yeast cells.
It is used for in vivo as well as in vitro gene therapy.
Yet another important benefit of it is, it can transfer any sized plasmid DNA into cells.
Broadly, the electroporation method is effective, reproducible, fast, efficient and easy to use. Importantly, the insertion is transient and stable.
Limitations:
Besides, the benefits of the electroporation technique, It has one critical limitation.
Under the influence of higher current or voltage, cells may die. The apoptosis rate using the electroporation method is very high.
Once the transformed cells are dead, it’s of no use.
Transfection: it’s a process of introducing nucleic acid into live eukaryotic cells.
Transformation: Direct uptake of exogenous nucleic acid through the cell membrane of bacteria, plant or yeast cells.
Applications:
It’s used in gene transfer and gene therapy experiments.
Foreign DNA, gene, viral DNA or plasmid DNA can be inserted into target cells using the present method.
It is effectively employed in knockout mice construction. Gene expression studies are done using the gene knockout or gene knock-in studies.
The electroporation technique is often used for cancer prevention. Anticancer agents or gene can be inserted in affected cells.
It’s used in vaccine development.
Furthermore, due to the high efficiency of the present method, it is used in drug discovery and drug research as well.
It is often used to induce a rapid and robust immune response.
The electroporation technique practised for generating monoclonal antibodies since long.
The present method is also useful in RNA interference studies, gene expression and gene silencing studies.
Broadly, the major applications of it are in farming, plant tissue culture, microbiology, medicines and molecular biology.
Apart from its major applications, the electroporation method is also widely used for transferring dye, drugs, protein, antibodies, peptides and chemicals into cells for various purposes.
Conclusion:
Although, the electroporation method is yet not fully ready for gene therapy. The present research indicates positive results. It is one of the effective and better ways of inserting anything into a cell.
However, the efficiency of gene transfer is far more suing viral-vectors.
Source:
Potter H. Transfection by electroporation. Curr Protoc Mol Biol. 2003; Chapter 9:Unit–9.3.
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