Transfection in vitroTransfection protocol Download
Transfection in vitro can be mediated by the non-viral reagents (such as polymer, liposome, nano-particle) or viral vectors (such as lentivirus, adenovirus and AAV).
Polymer transfection is a technique used to deliver DNA or RNA into cells with biodegradable cationic polymer , such as DEAE-dextran or polyethylenimine (PEI). The negatively charged DNA binds to the polycation and the complex is taken up by the cell via endocytosis. With some toxicity, polymer conjugated complexes can mediate transfection efficiently in some cell types, such as 293T cells, but are not suitable for transfection of sensitive cells and generation of stable cell lines.
Liposome transfection, also known as lipofection or lipid transfection, is a technology used to deliver DNA or RNA into cells mediated by liposomes, which are tiny vesicular structures with the same composition as the cell membrane and can easily fuse with the cell membrane . To date, there have been several generations of liposome, differing in the addition of cationic lipid and DNA-compacting substances. Now the most commonly used liposome is highly potent cationic lipofection reagent that has been shown to effectively transfect plasmids or siRNA, as well as nucleic acid-protein complexes, into cultured adherent and suspension cell lines . Besides, the efficiency of lipofection can be improved by treating transfected cells with a mild heat shock . Genemedi has launched a transfection reagent of Lipogene, which is comparable to Lipofectamine 3000, much more effective than polyethylenimine (PEI).
You could find more information on this website: www.genemedi.net/i/lipogene-transfection-reagent.
Non-viral gene delivery system – multifunctional envelope-type nano-devices (MEND) – has been proposed to package DNA or RNA into a nano-sized structure with programmed assembly of functional devices, enabling DNA protection from DNase, size control and improved packaging efficiency. Then polycations and lipids were added based on electrostatic interactions to realize lipid-film hydration . Now Impalefection has been developed to impale cells to deliver DNA or RNA using nanomaterials, such as carbon nanofibers, nanotubes, nanorods, silicon nanowires . Nano-particle transfection transfers the DNA or RNA via membrane fusion into the cell, avoiding DNA/RNA degradation by lysosome and showing great advantages over polymer and liposome.
Electroporation, also known as gene electrotransfer, is a popular method to realize high efficient transfection. During electroporation, cells and DNA/RNA of interest are exposed to short pulses of an intense electric field, leading to a transient increase in cell membrane permeability and the entry of DNA/RNA around into cytoplasm. Once removing the electric field, the cell membrane stabilizes and closes, allowing the expression of the enclosed DNA/RNA. Though electroporation-mediated transfection is easily operated and reliable, but it leads to high rates of cell death and requires great numbers of cells. Besides, a special and matched electroporation device is needed for electroporation .
|Principle||Endocytosis||Endocytosis||Membrane fusion/impale cells||Transient increase in the permeability of cell membrane|
|Time to peak expression||48h-72h||48h-72h||24h-48h||48h-72h|
|Sustainable time||Transient expression||Transient expression||Transient expression||Transient expression|
|Cell Type||A number of cell types||Adherent and suspension cell lines||Almost all the cells||Not suitable for sensitive cell types|
|Particle diametre||75 to 520 nm||50-200nm||100-300nm||-|
|Animal experiment||Low efficiency||Low efficiency||Target delivery||Target delivery|
|Efficiency||Low efficiency (<10%)||Depend on cell type||High efficiency||High efficiency|
|Price||Inexpensive||Medium||Expensive||Most expensive (electroporation device)|
Lentivirus-based gene transduction in vitro
Lentiviral vectors can mediate efficient transfection and long-term expression of exogenous genes in both dividing and non-dividing cells and have been widely used for gene overexpression, RNA interference, microRNA research and in vivo animal experiments.
More useful information about lentivirus can be found on this website: https://www.genemedi.net/i/lentivirus-packaging .
Adenovirus-based gene transduction in vitro
Based on human adenovirus type 5 (Ad5), recombinant adenovirus (Ad) a replication-defective adenoviral vector system, is widely used for gene delivery in most cell types, and Genemedi got a rich experience in adenovirus packaging, you could find more information on https://www.genemedi.net/i/adenovirus-packaging .
AAV-based gene transduction in vitro
As the most excellent gene therapy vector, recombinant AAV can mediate long-term stable expression of target genes in vitro/vivo with broad range of host and low immunity. For instance, AAV-DJ/8 was engineered via DNA family shuffling technology, displaying better in vitro transduction efficiencies compared to that of other wild type serotypes and higher infectivity rates in various cells and tissue types ; and AAV6 shows great transduction efficiency in CD4+ and CD8+ T cells . Genemedi is good at AAV production, you can find more information and protocols about AAV on this website: www.genemedi.net/i/aav-packaging .
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