What is transfection and how does it work?
· Transfection is a process by which foreign nucleic acids are delivered into a eukaryotic cell to modify the host cell’s genetic makeup ( Kim & Eberwine, 2010; Chow et al., 2016 ). For the past 30 years, transfection has gained increasing popularity due to its wide application for studying cellular processes and molecular mechanisms of ...
How does KIFC1 drive tumor malignancy?
Gastric cancer (GC) is one of the most aggressive malignant tumors with low early diagnosis and high metastasis. Despite progress in treatment, to combat this disease, a better understanding of the underlying mechanisms and novel therapeutic …
What is the best method for transfection?
Transfection is the process of introducing nucleic acids into eukaryotic cells by nonviral methods. Using various chemical or physical methods, this gene transfer technology enables the study of gene function and protein expression in a cellular environment. The development of reporter gene systems and selection methods for stable maintenance ...
Why is the efficiency of transfection in primary cells low?
· The transfection methods are broadly classified into three groups; biological, chemical, and physical. These methods have advanced to make it possible to deliver nucleic …
What is the role of KIF23?
KIF23, which belongs to the KIF family, plays a vital role in various cell processes, such as cytoplasm separation and axon elongation. Nowadays, KIF23 has been found to be highly expressed in multiple tumor tissues and cells, suggesting a potential link between KIF23 and tumorigenesis.
What is a KIF23?
KIF23, which belongs to the KIF family, …. Gastric cancer (GC) is one of the most aggressive malignant tumors with low early diagnosis and high metastasis. Despite progress in treatment, to combat this disease, a better understanding of the underlying mechanisms and novel therapeutic targets is needed. KIF23, which belongs to the KIF family, ….
Is KIF23 correlated with pTNM?
Herein, we reported that KIF23 expression was correlated with poor prognosis of gastric cancer and found an association between KIF23 and pTNM stage. An in vitro assay proved that the proliferation of gastric cancer cells was significantly inhibited, which is caused by KIF23 depletion.
What is the goal of long term transfection?
The goal of stable, long-term transfection is to isolate and propagate individual clones containing transfected DNA that has integrated into the cellular genome. Distinguishing nontransfected cells from those that have taken up exogenous DNA involves selective screening. This screening can be accomplished by drug selection when an appropriate drug-resistance marker is included in the transfected DNA. Alternatively, morphological transformation can be used as a selectable trait in certain cases. For example, bovine papilloma virus vectors produce a morphological change in transfected mouse CI127 cells (Sarver et al. 1981).
What is transfection in biology?
Transfection is the process of introducing nucleic acids into eukaryotic cells by nonviral methods. Using various chemical or physical methods, this gene transfer technology enables the study of gene function and protein expression in a cellular environment. The development of reporter gene systems and selection methods for stable maintenance and expression of transfected DNA have greatly expanded the applications for transfection. Assay-based reporter technology, together with the availability of a wide array of transfection reagents, provides the foundation to study mammalian promoter and enhancer sequences, trans -acting proteins such as transcription factors, mRNA processing, protein:protein interactions, translation and recombination events (Groskreutz and Schenborn, 1997).
How long does it take to transfect a cell?
For initial tests with liposomal reagents that require adding or replacing the medium, use a 1-hour transfection interval, and test transfection times of 30 minutes to 4 hours or even overnight, depending on the reagent used. Monitor cell morphology during the transfection interval, particularly when cells are maintained in serum-free medium, because some cell lines lose viability under these conditions.
What is the best concentration of plasmid DNA for transfection?
Suspend ethanol-precipitated DNA in sterile water or TE buffer to a final concentration of 0.2–1mg/ml. The optimal amount of DNA to use in the transfection will vary widely, depending on the type of DNA, transfection reagent, target cell line and number of cells.
What are the parameters that affect transfection efficiency?
With any transfection reagent or method, cell health, degree of confluency, number of passages, contamination, and DNA quality and quantity are important parameters that can greatly influence transfection efficiency. Note that with any transfection reagent or method used, some cell death will occur.
How long after transfection can you perform a reporter gene assay?
Generally, reporter gene assays are performed 1–3 days after transfection; the optimal time should be determined empirically. A functional test for the protein of interest, such as an enzymatic assay, may be another method to determine transfection success.
Can plasmid DNA be transfected?
Plasmid DNA is most commonly transfected into cells , but other macromolecules can be transferred as well. For example, DNA oligonucleotides, small interfering RNA (siRNA), proteins, and ribonucleoprotein complexes (RNPs) have been successfully introduced into cells via transfection methods (reviewed in Stewart et al. 2016). However, conditions that work for plasmid DNA transfer will likely need to be optimized when using other macromolecules. In all cases, the agent transfected needs to be of high quality and relatively pure. Nucleic acids should be free of proteins, other contaminating nucleic acids and chemicals (e.g., salts from oligo synthesis). Protein should be pure and in a solvent that is not detrimental to cell health.
What is the purpose of transfection?
Transfection is a powerful analytical tool enabling study of the function of genes and gene products in cells. The transfection methods are broadly classified into three groups; biological, chemical, and physical. These methods have advanced to make it possible to deliver nucleic acids to specific subcellular regions of cells by use of a precisely controlled laser-microcope system. The combination of point-directed transfection and mRNA transfection is a new way of studying the function of genes and gene products. However, each method has its own advantages and disadvantages so the optimum method depends on experimental design and objective.
How will transfection technology change the future?
axon or dendrite) and organelles (e.g. mitochondria, golgi apparatus, or nucleus) will open an new era for genetic research because it will change how we think about and assess the function of genes in a cell. In addition to the overall gene expression profiles of a cell, the location of expressed gene products plays a crucial role in determining the function of a cell [20]. Meanwhile, safe and reliable transfection methods that can be applicable to humans are needed to establish clinical therapeutics.
How long does it take for mRNA to be expressed after transfection?
transfected mRNAs can be expressed within minutes after transfection because it skips translocation to the nucleus and transcription process; and
Why is mRNA transfected?
Transfecting mRNA has several merits over DNA transfection [26]. The merits include no risk of integration into the host genome, cell cycle- independent transfection efficiency , no need for immune inducible vectors, and adjustable and rapid expression . Using mRNA transfection, one can introduce any number of mRNAs into a cell, thereby overcoming overexpression of the genes. These advantages mostly originate from the fact that mRNA does not need to be located in a nucleus to be expressed. Transfected DNA must carry a host cell or tissue-specific promoter to be transcribed to mRNA and the expression level is determined by strength of the promoter. In contrast with DNA transfection, one can adjust expression levels by changing the amount of mRNA transfected and the frequency of transfection in mRNA transfection. Other strong advantages of mRNA transfection are:
How do you deliver nucleic acids?
The physical transfection methods are the most recent methods and use diverse physical tools to deliver nucleic acids. The methods include direct micro injection, biolistic particle delivery, electroporation, and laser-based transfection [13]. In brief, the micro injection method directly injects nucleic acid into the cytoplasm or nucleus [14, 15]. This method delivers nucleic acids into cells but demands skill, often causes cell death, and is very labor-intensive. Biolistic particle delivery employs gold particles that conjugate with nucleic acids [16, 17]. The nucleic acid/particle conjugates are then shot into recipient cells at a high velocity (“gene gun”). This method is straightforward and reliable but it requires expensive instruments and causes physical damage to samples. Electroporation is the most widely used physical method. The exact mechanism is unknown but it is supposed that a short electrical pulse disturbs cell membranes and makes holes in the membrane through which nucleic acids can pass [18]. Because electroporation is easy and rapid, it is able to transfect a large number of cells in a short time once optimum electroporation conditions are determined. Laser-mediated transfection (also known as optoporation or phototransfection) uses a pulse laser to irradiate a cell membrane to form a transient pore [19–22]. When the laser induces a pore in the membrane, nucleic acids in the medium are transferred into the cell because of the osmotic difference between the medium and the cytosol. The laser method enables one to observe the transfecting cell and to make pores at any location on the cell. This method can be applied to very small cells, because it uses a laser, but it requires an expensive laser-microscope system. In addition to those mentioned above, there are other physical methods using ultrasound (sonoporation) and magnetic field (magnetofection) [23–25].
What are the most common methods of transfection?
Chemical transfection methods are the most widely used methods in contemporary research and were the first to be used to introduce foreign genes into mammalian cells [10]. Chemical methods commonly use cationic polymer (one of the oldest chemicals used), calcium phosphate, cationic lipid (the most popular method), and cationic amino acid [10–12]. The underlying principle of chemical methods is similar. Positively charged chemicals make nucleic acid/chemical complexes with negatively charged nucleic acids. These positively charged nucleic acid/chemical complexes are attracted to the negatively charged cell membrane. The exact mechanism of how nucleic acid/chemical complexes pass through the cell membrane is unknown but it is believed that endocytosis and phagocytosis are involved in the process. Transfected DNA must be delivered to the nucleus to be expressed and again the translocation mechanism to the nucleus is not known.
What are the drawbacks of virus-mediated transfection?
The major drawbacks of virus-mediated transfection are immunogenicity and cytotoxicity. Introduction of a viral vector may cause an inflammatory reaction and an insertional mutation, because viral vectors integrate into the host genome randomly, which may disrupt tumor suppressor genes, activate oncogenes, or interrupt essential genes [9]. Another disadvantage of this method is that a virus package has limited space for a foreign gene to keep infectivity. For these reasons, much effort has been made to develop non-viral transfection methods even though virus-mediated transfection is highly effective and easy to use.
Is KIFC1 essential for somatic cells?
In this regard, KIFC1 seems to be non-essential in normal somatic cells which usually possess only two centrosomes. Moreover, KIFC1 …. The kinesin motor KIFC1 has been suggested as a potential chemotherapy target due to its critical role in clustering of the multiple centrosomes found in cancer cells.
Is KIFC1 a target for chemotherapy?
The kinesin motor KIFC1 has been suggested as a potential chemotherapy target due to its critical role in clustering of the multiple centrosomes found in cancer cells. In this regard, KIFC1 seems to be non-essential in normal somatic cells which usually possess only two centrosomes.
What are the applications of transfection?
Transfection has many applications. Besides basic research, these include gene therapy, immunotherapy and induced pluripotent stem cell generation. Meanwhile, recombinant protein expression also enables a wide range of functional, genetic mapping and biochemical characterization studies, in addition to bioproduction applications.
What is the purpose of transfection?
Transfection is generally defined as the process of introducing DNA, RNA or proteins into cells to influence their genotype or phenotype. This includes introducing new genes to study or leverage their function, as well as using other constructs to indirectly influence endogenous gene expression ...
Why are proteins important in transfection?
The advantage of using proteins is that they usually exert their influence on the cell immediately and the quantity can be controlled much better. However, proteins vary greatly in terms of size, shape and charge, so transfection conditions must often be specifically optimized on a per-protein basis.
How to use plasmid DNA?
Plasmid DNA is commonly used for transfection (although linear DNA can also be used). In both cases, you should ensure that your DNA is as pure as possible, with any contaminating lipids, salts, proteins, nucleotides or other factors removed via DNA purification. The best way to obtain high quality DNA is by using a purification method based on anion exchange technology which includes an additional endotoxin removal step. This helps to ensure optimum transfection efficiency and cell viability. When transfecting DNA purified with silica membranes, there is a high risk, on poor transfection efficiency and cell viability, further downstream results might not be trustable.
Can you transfect cells with DNA?
You can use DNA, RNA or proteins to transfect your cells, and each has its own advantages and disadvantages. Additionally, certain cell types or applications may only work with a specific transfection substrate, and you will often need to reoptimize your transfection conditions when switching substrates.
Why is stable transfection important?
Stable transfection is often required for large-scale protein production, research into long-term gene regulation, the generation of stable cell lines, and for gene therapy.
How to achieve stable transfection?
To achieve stable transfection, you must use genetic constructs that will be incorporated into the genome of the host cells (either into the chromosomes themselves, or as extra-chromosomal episomes), so that they will pass these new genes onto their offspring . The constructs must also carry selection markers, so that you can identify which cells have been successfully transfected. Stable transfection is often required for large-scale protein production, research into long-term gene regulation, the generation of stable cell lines, and for gene therapy. As the method requires successful DNA integration into the host genome, it is often much harder to achieve than transient transfection, and typically has lower transfection efficiency. Stable integration can occur randomly with plasmids, actively at random sites with help of transposases or viruses, or site-specifically when using genome editing tools like CRISPR.
What are the main modes of therapeutically relevant transfection?
Together, the companies mentioned thus far offer technologies that cover all the main modes of therapeutically relevant transfection: chemical-based transfection, nonchemical methods (chiefly electroporation), and viral transduction. Hybrid approaches, too, are being developed, as will become clear in the following sections.
What is electroporation in a cell?
Electroporation, the nonviral approach employed by MaxCyte, allows the biology of the transfected cells to remain in a more natural state at any scale. By preventing unintended consequences during the transfection process, MaxCyte’s technology can avoid creating roadblocks while solving the need for high transfection efficiency.
Is a 4D nucleofector scalable?
The Nucleofection platform is scalable. For example, the high-throughput Nucleofection system can handle 96-well and 384-well formats. Often this system will be employed in a core screening facility. Scientists at the bench can use the lower-throughput 4D Nucleofector to optimize assay conditions. Because the benchtop 4D Nucleofector uses the same transfection conditions, works with the same cell numbers, and delivers the same performance as the higher throughput devices, when it is time to move up to a larger scale, the assay does not require re-optimization.
What is the best substrate for nucleofection?
The Nucleofection approach utilizes many different substrates: DNA, mRNA, siRNA, peptides, proteins, and small molecules. Transfection efficiencies for siRNAs and mRNAs are very good, better than 90%. Small peptides usually transfect with around 80% efficiency, and efficiencies for plasmid DNA are anywhere from 50% to 90%, depending on cell type. Even large substrates, larger proteins such as antibodies or bacterial artificial chromosomes (BACs), can enter the target cells at reasonably effective transfection efficiencies.
Does GenVec have a cell line?
“When the time comes to commercialize a technology, GenVec uses a cell line previously approved by the FDA for these applications,” clarifies Dr. Brough.
Can genvec be used in vitro?
By utilizing a variety of approaches, GenVec has worked with a number of companies on different applications, nucleic acid therapeutics, and gene-editing technologies. Modified adenoviruses can deliver zinc finger approaches, both in vitro and in vivo.
Is transfecting cells a chemical or lipid?
While chemical and lipid approaches work well in research with established cell lines, there are often unanticipated consequences of transfecting primary cells. Additionally, these traditional chemical means of transfecting cells are difficult to scale up to industrial production.
What happens if you plated a plate before transfection?
a. If plated >1 day prior to transfection, the transfection efficiency may decrease.
What reagent to use for passage cells?
i. Passage cells at or before 90% confluency. We recommend using Gibco™ TrypLE™ reagent for detachment of cells. TrypLE reagent can be stored in the hood at room temperature, and you can skip the PBS wash by adding extra TrypLE reagent and aspirating all but enough solution to cover the surface of the flask.
How long to incubate lipids in Opti-MEM?
c. Once the lipid is diluted in Opti-MEM medium, the optimal incubation time is 2 to 5 min before adding the diluted DNA. Do not incubate the diluted lipid for longer than 20 min.
Can you do cell plating and transfection at the same time?
Cell plating and transfection can be performed at the same time or by using a reverse transfection protocol. In a reverse transfection, you need to use 2.5 times more cells than you would use in a regular or forward transfection. e.
How many times should you thaw passage cells?
a. Passage cells 3–4 times after thawing before using them in transfection experiments. This gives the cells time to recover from the thawing procedure and return to their normal rate of growth.
Can transfection complexes be added to cells in media containing antibiotics and serum without impacting the transfection
e. Transfection complexes can be added to cells in media containing antibiotics and serum without impacting the transfection efficiency.
What reagent is used to transfect 17 cell lines with a GFP-expressing plasmid?
Figure 6.1 Cell line-dependent differences in transfection efficiency. Invitrogen product line such as Lipofectamine 2000 reagent and Lipofectamine 3000 reagent were used to transfect 17 cell lines with a GFP-expressing plasmid in a 24-well plate format, using 0.5 µg plasmid/well and the recommended protocols for each reagent. GFP expression was analyzed 48 hours posttransfection. Each condition was tested in triplicate, and the data points show the mean transfection efficiency plus standard deviation.
How long before transfection should cells be subculturing?
We strongly recommend subculturing cells at least 24 hours before transfection to ensure that they recover from the subculture procedure and are in optimum physiological condition for transfection.
How long can cells stay healthy in serum free medium?
Most cells remain healthy for several hours in a serum-free medium. The quality of serum can significantly affect cell growth and transfection result. Therefore, it is important to control for variability among different brands or even different lots of serum to obtain best results.
Why is serum important in cationic transfection?
However, when performing cationic lipid-mediated transfection, it is important to form DNA-lipid complexes in the absence of serum because some serum proteins interfere with complex formation.
What happens if you have too many cells in a culture?
However, too few cells in culture may result in poor growth without cell-to-cell contact. In such cases, increasing the number of cells in culture improves the transfection efficiency.
How long can cells stay confluent?
Do not allow the cells to remain confluent for more than 24 hours. The optimal cell density for transfection varies for different cell types, applications, and transfection technology, and should be determined for every new cell line to be transfected.
How does passaging affect transfection?
Excessive passaging is likely to detrimentally affect transfection efficiency as well as total transgene expression level from the cell population as a whole.
Why is T-cell transfer therapy used?
T-cell transfer therapy was first studied for the treatment of metastatic melanoma because melanomas often cause a strong immune response and often have many TILs. The use of TIL therapy has been effective for some people with melanoma and has produced promising findings in other cancers, such as cervical squamous cell carcinoma and cholangiocarcinoma. However, this treatment is still experimental.
What is T cell transfer?
T-cell transfer therapy is a type of immunotherapy that makes your own immune cells better able to attack cancer. There are two main types of T-cell transfer therapy: tumor-infiltrating lymphocytes (or TIL) therapy and CAR T-cell therapy.
Can car T cells cause organ damage?
Also, although CAR T cells are designed to recognize proteins that are found only on cancer cells, they can also sometimes recognize normal cells. Depending on which normal cells are recognized, this can cause a range of side effects, including organ damage. TIL therapy can cause capillary leak syndrome.
Can cytokine release syndrome be life threatening?
Most people have a mild form of cytokine release syndrome. But in some people, it may be severe or life-threatening.
What happens when T cells are transferred to a new T cell?
This syndrome is caused when the transferred T cells, or other immune cells responding to the new T cells, release a large amount of cytokines into the blood. Cytokines are immune substances that have many different functions in the body.
Is car T cell therapy approved for cancer?
Three CAR T-cell therapies have been approved by the Food and Drug Administration for blood cancers: CAR T-cell therapy has also been studied for the treatment of solid tumors, including breast and brain cancers, but use in such cancers is still experimental.
Is til therapy effective for melanoma?
The use of TIL therapy has been effective for some people with melanoma and has produced promising findings in other cancers, such as cervical squamous cell carcinoma and cholangiocarcinoma. However, this treatment is still experimental.