Transfection Methods and Techniques

Transfection Methods

The introduction of DNA and RNA molecules into cultured mammalian cells requires the use of various transfection methods that depend, in part, on the cell lines being utilized and the types of experiments being performed. These method, discussed in more detail below, include chemical (liposome-mediated, non-liposomal lipids, polyamines, dendrimers), physical (electroporation, microinjection, heat shock), or viral-based (retrovirus, adeno-associated virus, lentivirus) delivery systems.

Liposome-mediated transfection
Liposomes are synthetic analogues of the phospholipid bilayer of the cellular membrane. These compounds contain a number of the physical characteristics of phospholipids including the presence of hydrophobic and hydrophilic regions of each molecule which allows for the formation of spheroid liposomes under aqueous conditions. In the presence of DNA or RNA, liposomes are capable of interacting with and encapsulating the nucleic acids thereby creating an efficient delivery system. The liposomal charge, composition and structure, defines the affinity of the complex for the cellular membrane. Under specific conditions, the liposome complex is able to interact with the cell membrane, which enables its uptake by endocytosis and subsequent release into the cellular cytoplasm. The successful use of liposome compounds to deliver exogenous nucleic acids to specific biological system is dependent upon several factors including lipid formulation, charge ratio, particle size and the method of liposome preparation.

Non-liposomal transfection agents (lipids and polymers)
Several non-liposomal lipids and some polymers have been developed that are capable of forming complexes with DNA or RNA and have the potential to form micelles. The transfection reaction is usually performed under aqueous conditions which enables the lipophilic portion of the amphiphilic compound to form the micelle core within which the exogenous nucleic acids are ensconced. Superior transfection efficiencies can be achieved in cell lines that are refractory to liposome-based transfection.

Dendrimer-based transfection
Dendrimers are highly branched, globular macromolecules that are capable of interacting with and condensing DNA in small complexes. Dendrimers are typically stable in serum and are not temperature sensitive. Therefore, it is believed that these characteristics of dendrimers are responsible for the high plasmid transfection efficiencies observed in several tissue culture models. However, dendrimers are non-biodegradable and at lower than expected concentrations may cause significant cellular toxicity thereby inadvertently affecting the outcome of the experiment.

Electroporation is a highly efficient technique, and often the only option, for delivering exogenous nucleic acids to cells grown in suspension and certain primary cells. This technique employs the use of an electrical field to create transient pores, known as electropores, in the cellular membrane which enables the delivery of charged molecules like RNA or DNA to the cytoplasm and nuclei of the targeted cells.

Certain cell types and/ or experimental conditions require that specific cells within a population are targeted for gene delivery. In these instances gene guns or manual microinjection are very efficient techniques for the direct delivery of DNA to specified target cells. However, the method is limited with regard to the number of cells to which the exogenous nucleic acids can effectively be delivered and requires certain operator skills.

Virus-mediated gene delivery
DNA can be introduced into cultured mammalian cells by viral transduction technique using viruses as carriers. Viral delivery is beneficial for transfection of primary cell cultures and numerous studies have developed in vivo gene delivery approaches, however the clinical and laboratory uses of these techniques also carry significant bio-hazardous risks.

In Vivo Transfection Kits from Altogen Biosystems

RNAi has been used for in vivo target validation studies using animal models. The major challenge in performing RNAi studies in vivo is the effective, directed delivery of functional siRNA, shRNA, and miRNA molecules into specific tissues. ALTOGEN® In Vivo Transfection Reagents could be conjugated with siRNA and administered intratumorally (i.t) or systemically via intravenous (i.v) tail vein injection in order to provide directed gene silencing in specific tissues, including liver, pancreas, kidney, and tumors. Selective knockdown could be seen as early as 24 hours after injection.

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Transfection Services by Altogen Labs CRO:

Altogen Labs CRO offers biotechnology and pharmaceutical contract research services, including: in vivo toxicology, xenograft services, generation of stable cell lines, RNA interference (RNAi) services, pharmacology and toxicology testing: IC-50, ELISA assay development, siRNA library screening and transfection services, bioremediation products and services, cell banking and cryopreservation services.

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Altogen Biosystems is a life sciences company dedicated to the development, marketing and manufacture of cell type specific transfection reagents. Efficient delivery of DNA, RNA, and siRNA enabled by advanced formulation of reagents and peculiar design of protocols. Altogen Biosystems offers a complete transfection system for a broad range of cell lines. All reagents are functionally tested to be highly reproducible, serum compatible, induce low toxicity, and can be used for co-transfection experiments, and high throughput applications.