Introduction to transfection
Transfection is a process involving introduction of foreign nucleic acids into eukaryotic cells, resulting in modification of host cell genome. The modification can take place either through expression of exogenous genetic material or by means of endogenous gene silencing. Transfection is a powerful tool for studying cell physiology and complex molecular mechanisms of diseases, as it can shed more light on gene regulation and protein function1. Various types of nucleic acids can be transfected into mammalian cells, including plasmid DNA (pDNA) and small, non-coding RNAs, such as small interfering RNA (siRNA), microRNA (miRNA), and short hairpin RNA (shRNA)2. Intact functional proteins (e.g. Cas9, recombinant proteins, antibodies) and peptides can also be directly delivered into cells, offering significant potential for clinical applications3. Broadly, the transfection methods can be subdivided into biological (e.g. transduction), physical (e.g. electroporation, microinjection), and chemical (e.g. lipofection, calcium phosphate transfection) methods1.
Transduction, also known as virus-mediated transfection, employs viral vectors, such as lentiviruses and retroviruses, for delivery of nucleic acids. Viral transfection remains as one of the most commonly used methods, due to the inherent capabilities of viruses to gain access to host cells, resulting in sufficiently high transfection efficiency 4,5. However, there are some issues associated with the use of viral carriers, including immunogenicity, cytotoxicity, and oncogenic potential6. A physical/mechanical approach involves forceful injection of nucleic acids, without any carrier, into the cytosol or nucleus of a host cell. For instance, electroporation is routinely used for delivering nucleic acids and it relies on high-intensity electrical pulses that cause transient and localized destabilization of the cell membrane7. Lastly, chemical methods utilize the ability of positively charged chemical vectors and negatively changed nucleic acids to form nucleic acid-chemical vector complexes. Since the resulting charge of these complexes remains positive, it makes them attracted to the negatively charged membrane of a cell. Multiple factors have to be considered before choosing a suitable transfection protocol, including the goal of the experiment, cell type, and the nature of transfected molecules. Regardless which type of transfection is chosen, the ideal method should result in high transfection efficiency, minimal effects on cell viability and physiology, while also being easy to perform and reproduce1.
Evaluation of transfection efficiency
Transfection efficiency is the proportion of cells in a sample that acquired a foreign element and it can be assessed using a number of different approaches. Firstly, transfection efficiency can be confirmed and quantified through the analysis of target gene or protein expression post-transfection. This method evaluates total gene/protein expression from a transfected cell population and it can be measured through e.g. western blotting, real-time quantitative PCR (real-time qPCR), reporter system, or molecular imaging. Transfection efficiency can also be evaluated by determining the proportion of transfected cells within a total cell population. This can be done by coupling a transfected gene with a fluorescent reporter (see: Lux3 FL compatible protein list), either by placing them on separate delivery vectors or by introducing them together, through a transcriptional/translational reporter gene fusion8. Subsequently, transfection efficiency can be assessed by estimating the number of fluorescent cells either using flow cytometry or fluorescence microscopy9.
Lux3 FL: real-time analysis of transfection efficiency
Live-cell, time-lapse imaging is a powerful approach that can be used for monitoring the time course of transfection, providing a valuable, time-dependent insight into the process10. Compared to other methods of transfection efficiency analysis (i.e. western blotting, flow cytometry, or real-time qPCR), live-cell imaging and microscopy in general offer a more straightforward and direct route to evaluating the efficiency of foreign molecule uptake. However, even among different live-cell imaging modalities, there are approaches capable of providing reliable results quickly and easily, while requiring minimal expenses.
The CytoSMART Lux3 FL combines brightfield and fluorescence imaging to directly visualize and monitor the uptake and expression of foreign elements by transfected cells (Figure 1). The device can also be used for evaluating the efficiency of transfection based on the fluorescence surface area or the number of fluorescent objects in the image:
- Surface area: transfection efficiency can be calculated as a ratio of the surface area occupied by fluorescent cells to the total surface area of cells, as determined by the brightfield channel
- Object count: while transfection efficiency cannot be measured directly using the integrated Object Count algorithm, the CytoSMART Lux3 FL can calculate the number of fluorescent objects in the image and track fluorescent object number over time
Monitoring the process of transfection not only allows to examine transfection-induced changes in gene expression and notice trends that otherwise might have been missed, but it can also assist in optimization of a transfection protocol. Viability and density (confluency) of cells are known factors that influence transfection efficiency. Live-cell imaging can be used to assess cell health and confluency prior to and during transfection to maximize the efficiency of nucleic acid uptake.
Figure 1. Time-lapse video of HepG2 cells transduced using the BacMam System. HepG2 cells were transduced in suspension using CellLight Nucleus-RFP BacMam 2.0 and CellLight Actin-GFP BacMam 2.0 reagents for fluorescent detection of nuclei (red signals) and filamentous actin (green signals). Images were taken using the CytoSMART Lux3 FL with a snapshot interval of 30 min.
Exact FL: endpoint analysis of transfection efficiency
The CytoSMART Exact FL is an automated, dual fluorescence cell counter with an expanded field of view and increased optical resolution that provides accurate and reliable analysis of transfection efficiency. Similarly to other microscopy-based approaches, the Exact FL directly visualizes transfected, fluorescently-labeled cells in a sample. However, unlike the CytoSMART Lux3 FL, it does not generate time-lapse videos of cell cultures. To measure transfection efficiency, the cells transfected with a construct containing a reporter gene, such as gfp, are loaded into reusable/disposable counting chamber. Next, the sample is placed on the cell counter stage and the device captures high-resolution images of the cells. The images are automatically processed using AI-based image analysis software to quantify the proportion of cells that have successfully acquired a transfected vector. The ease of use, accuracy, and reduced user-to-user variability in the assessment of cell numbers make the CytoSMART Exact FL an ideal tool for endpoint analysis of transfection efficiency.
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