Transfection is the use of chemical (liposomes, chemicals) or mechanical (electroporation) means to transfer nucleic acids into cells in order to alter mRNA expression or the genetic makeup of a genome (i.e. stable expression of exogenous genes or gene knockout).
Non-viral delivery of DNA or RNA requires a transfection reagent or transfection method with multi-faceted capabilities. Attributes of a good transfection reagent include protecting the payload from harsh environments that can lead to degradation from RNases and DNases by forming a tight bilayer (hydrophobic exterior, hydrophilic interior). A successful transfection reagent also readily loads with a high ratio of active pharmaceutical ingredient (API) to delivery agent ratio. This ultimately leads to lower manufacturing costs of the transfection reagent since lower concentrations can be used per test sample. Perhaps the most crucial aspect of a transfection reagent is size. With an understanding of cellular pore size and an extensive innate response to foreign material in an organism, transfection reagents must have final structure sizes that can bypass mechanisms that trigger immune responses. Optimal transfection reagent compound size should fall in a range of 50-400 nm.
As noted, well-constructed transfection reagents offer compact, protective delivery vehicles capable of carrying a high ratio of DNA or RNA cargo and can deliver the payload to the desired cells/tissues without being toxic to the cells. Transfection is a highly… [Read more]
In Vitro Lipid Transfection
Manipulation of gene expression by transfection of cultured cells enables a fully controlled, clean environment for the study of apoptosis, cell cycle, target gene expression and mechanism of action. There is a vast array of in vitro transfection reagents available on the market that is specific to the researchers needs. Although cell culture studies do not require a transfection reagent to protect the payload from the harsh environment of circulation ion animals, it must provide protection from degrading factors in cell culture medium FBS serum (i.e. DNases and RNases).
- In Vitro Transfection Reagents- Cell Line Specific
Cells that remain difficult to transfect using lipid based reagents are often transfected via electroporation. Electroporation is the use of electrical pulses to permeabilize (form small pores) in a cell membrane. Along with pore formation, the transfection by electroporation electrical pulse physically moves nucleic acid (siRNA, miRNA, DNA) with the current and by chance, forces it into a cell’s cytoplasm. This is a physical means to transfect a cell and is highly toxic; thus, electroporation buffers are available that provide a healing environment to lessen the transfection effect.
- Electroporation Transfection Kit
- Electroporation Buffers are suitable for:
In Vivo Transfection
Exogenous nucleic acids delivered by transfection in an animal can be categorized in two ways, dependent on delivery of the payload to be specific or broad. Delivery to specific cells or organs is termed a “tissue-targeted” transfection reagent, whereas non-specific payload delivery is termed a “broad range” transfection delivery reagent. The differences in these two types of delivery is related to moieties and design of the transfection reagent. Broad delivery is a result of a transfection reagent fusing with the cells lipid bilayer in a non-specific matter. On the other hand, targeting cell surface markers using conjugates leads to transfection reagent cell specific delivery.
Due to the harsh environment in circulation, the nucleic acid cargo must be protected from clearance and degradation by the delivery agent, along with not triggering an immune response.
- In vivo PEGylated Liposome
- In vivo Nanoparticle Transfection Reagent
- LIPID In vivo Transfection Reagent
- Others here
As with any well designed experiment, both positive and negative controls need to be included in a transfection study design. Whether looking at multiple gene target expression knockdown using miRNA or a single target such as luciferase expression, the appropriate transfection controls are needed. The use of controls must be included whether you use a lipid transfection reagent or electroporation.
Transfection positive controls typically consist of siRNAs designed against gene targets that are known to be easily targeted and typically exhibit a large decrease in expression. Positive control siRNAs used in transfections include gene targets such as GAPDH, KIF11 or PPIA. The results of the transfected positive control siRNA tell the researcher the system works and is able to deliver the payload to the cells, and the end-point readout exhibits a quantitative signal that is significantly separated from the non-treated or negative control sample.
Transfection negative controls are nucleic acids that are similar in length and structure to the siRNA being tested. However, the negative control siRNA sequence is designed such that it is not complimentary to any known genes and is commonly called an alien sequence. Upon transfection, the negative control siRNA does not bind to any gene targets, thus gene expression levels, cell cycle and proliferation are not altered and mimic levels of the non-treated sample.
Stable Expressing Cells
Generation of a stable cell line is a two-step procedure, the first being to transfect exogenous nucleic acids into a cell or silencing a particular gene (knockout). This is followed by application of antibiotics to select cells exhibiting desired expression. Nucleic acids to be inserted can be in the form of plasmid DNA, and/or encoding microRNA (miRNA) or short interfering RNA (siRNA).