Transfection is a powerful technique used in genetic research to introduce foreign nucleic acids into eukaryotic cells to modify the genetic makeup of the host cell. This technique plays a crucial role in the study of gene function and the manipulation of gene expression. In addition, transfection can be used as a strategy for gene therapy to treat genetic diseases. By transferring exogenous genetic material (e.g. DNA, RNA, siRNA, shRNA and miRNA) into target cells, scientists can study various cellular processes and develop new therapeutic approaches for genetic diseases.
Transient or Stable Transfection?
There are two main transfection methods: transient transfection and stable transfection. The choice between transient or stable transfection usually depends on the experimental target and cell type.
In transiently transfected cells, the exogenous genes are expressed but they are not integrated into the cell's genome and are not replicated. Transiently transfected exogenous gene expression in cells has a limited time, usually only for a few days until the exogenous gene is lost due to various factors during cell division.
Stable transfection is based on transient transfection. Cells are transfected first, and then the obtained cell pool is screened. In a small number of transfected cells, exogenous genes can be integrated into the genome of the cells. A hallmark of a stably transfected cell is that the foreign gene becomes part of the cell's genome and thus replicates. Progeny cells of the stably transfected cells also express the foreign gene, thereby forming a stably transfected cell line.
| Differentiation |
Transient Transfection |
Stable Transfection |
| Mode of exogenous gene integration |
The introduced foreign gene will not be integrated into the host chromosome and will not be passed on to offspring |
The introduced foreign DNA is integrated into the genome and can be passed on to offspring |
| Type of exogenous gene |
Suitable for both DNA vectors and RNA |
Only DNA vectors are suitable, and RNA itself cannot be stably introduced into cells |
| Copy number |
High copy number results in high levels of protein expression |
Single copy number or low copy number stable integration results in lower levels of protein expression |
| Screening marker |
No selective screening required |
Antibiotics, fluorescent reporter groups, etc. |
| Duration of transfection |
Perform assays within 24-96 h of transfection |
2~3 weeks to screen stable cell lines |
| Expression duration |
Dilution to loss with cell division (gradual loss after 96 hours) |
Replicate, transcribe, translate, and be stably inherited along with the host cell's own genome |
| Transfection target |
Quick analysis |
Obtain single-cell clones stably expressing foreign genes |
Assessing Transfection Efficiency
Transfection efficiency is defined as the percentage of cells that successfully take up and express the introduced genetic material. Accurate assessment of transfection efficiency is essential for researchers to assess the reliability of their experimental results, particularly where high transfection efficiency is required to ensure the post-transcriptional regulation of a specific downstream target. A variety of approaches can be used to measure transfection efficiency, each of which has different advantages and disadvantages.
| |
Fluorescence Microscopy |
Real Time PCR (qPCR) |
Plasmid Reporting System |
Flow Cytometry |
| Descriptions |
A powerful technique that allows for the rapid analysis of individual cells based on their fluorescence properties |
Direct measurement of nucleic acid expression levels after transfection |
Indirect measurement of transfection efficiency by luminescence measurements or β-galactosidase levels |
Quantify the number of fluorescently labeled transfected cells |
| Quantitative or qualitative |
Qualitative or semi- and quantitative |
Quantitative |
Quantitative using luminescence reporting system |
Quantitative |
| Advantages |
Easy and fast |
Allows quantification of the transfection efficiency |
Allows quantification of the transfection efficiency |
Allows quantification of the transfection efficiency |
| Disadvantages |
Inability to
distinguish signals originating from interior vs exterior of cells |
Expensive and laborious especially in transient transfection where regular monitoring of the transfection efficiency is needed |
Reporting system within plasmids not offered by all manufacturers |
Expensive, and laborious |
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