What is Transfection?
Transfection is the transient or stable introduction of exogenous molecules and genetic material, DNA or RNA, into cultured mammalian cells and is commonly utilized in biological laboratories for studying gene function, modulation of gene expression, biochemical mapping, mutational analysis, and protein production. Researchers use various carrier molecules to enable non-viral gene delivery of plasmid DNA (pDNA), messenger RNA (mRNA), short interfering RNA (siRNA), and microRNA (miRNA) into cancer cell lines and primary cells. Unfortunately, no single delivery method or transfection reagent can be applied to all types of cells; cellular cytotoxicity and transfection efficiencies vary dramatically depending on the reagent, protocol and cell type being utilized.
History and Development
Transfection by calcium phosphate was one of the earliest chemical based methods developed to deliver exogenous nucleic acids to cultured mammalian cells. This method is dependent upon the use of a calcium chloride-containing HEPES-buffered saline solution to deliver plasmid DNA. Advances in the development of lipid and polymer-based carrier molecules capable of binding nucleic acids led to the adaptation of these compounds for transfection. Many of these compounds create liposomes, which can fuse with the cellular membrane in order to deliver the bound RNA or DNA to the cell. Recently, non-liposomal lipids and organic compounds called dendrimers have been developed and applied as a transfection agent for use with difficult to transfect cell lines. The in vitro uses of these methods are a powerful tool in basic life sciences research and associated clinical studies.
Methods of Transfection
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.
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.
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.
Transient and Stable Transfection
Transfected genetic material can be expressed in the target cells either transiently or permanently depending on the methods utilized and the experimental questions being investigated. Transient transfections are used most commonly to analyze the short term impact of altered gene or protein expression. Plasmid DNA (pDNA), messenger RNA (mRNA), short interfering RNA (siRNA), and microRNA (miRNA), are introduced and gene products are expressed in the target cells however the nucleic acids do not integrate into the host cell genome. Therefore, gene product expression is transient and typically results in high expression levels that persist for 24-72 hours when RNA is transfected, or 48-96 hours following DNA transfection. Conversely, in order to analyze the long term impact of altered gene or protein expression investigators typically utilize stable transfection protocols to develop stable cell lines. In a subpopulation of transfected cells, whether the desired effect is a stable or transient transfection, the transfected genetic material will integrate into the genome. In order to create stable cell lines, investigators will take advantage of this natural occurrence, and introduce the gene of interest along with a selectable marker. Therefore growth of transfected cells, in the presence of a selecting agent, will enable the subpopulation of cell where the exogenous genetic material has been incorporated into the genome to persist while the remaining cells undergo selection. Utilizing this method, investigators are able to develop cells that permanently express specific genes through their incorporation in the cellular genome.
Cancer Cell Lines and Primary Cells
Primary cell cultures are used in biological and gene therapy studies and serve as important model systems that may more accurately represent the biology of normal cells. Many cultured cell lines, as well as the majority of primary cell cultures are able to be transfected with exogenous nucleic acids when appropriate transfection approaches are employed. Since the majority of transfection methods causes significant toxicity in primary cell cultures, optimizing this procedure, specifically the protocol and reagents to be utilized, is essential for developing an effective transfection strategies for a given cell type.
RNA Interference (RNAi)
RNA interference (RNAi) is a phenomenon by which the expression of double stranded RNA (dsRNA) specifically stimulates a cellular process that reduces gene expression in a sequence specific manner. Small synthetic RNA, termed small interfering RNAs (siRNA), which are typically 21-28 nucleotides in length, can induce RNAi and knockdown gene expression in mammalian cells without inducing an antiviral response.
Biochemical studies have elucidated the mechanism of RNAi. Double stranded RNA is processed by an enzyme known as Dicer resulting in the production of siRNA molecules. These molecules are then able to form a multi-protein siRNA complex, known as the RNA-induced Silencing Complex (RISC). The RISC/siRNA complex catalyzes the cleavage and degradation of the complement mRNA molecules.
The use of RNAi applications, including small interfering RNA, to reduce cellular gene expression appears to have significant advantages over other approaches for targeted gene regulation. The potency of siRNA molecules, sequence-specific design, and ability of siRNAs to be re-used to guide mRNA degradation in cells bears a significant advantage over anti-sense oligonucleotides and ribozyme-stem approaches. Well-designed and functional siRNAs are capable of effectively bypassing the interferon response in order to induce specific post-transcriptional gene silencing or RNAi in vitro and in vivo.
Gene silencing by RNA Interference (RNAi) is a powerful research tool for studying gene function in mammalian cells. RNAi is a biological phenomenon by which double stranded RNA (dsRNA) specifically reduces gene expression of its corresponding gene. Potent inhibition of specific gene expression is experimentally achieved by the transfection of small interfering RNA (siRNA), which binds RISC complex and cause degradation of target complementary mRNA molecules in the cell. Therefore, successful, potent RNAi experimentation is dependent upon the highly efficient delivery of the siRNA into cells by transfection of stable and functional siRNA molecules.
Most DNA transfection reagents are incompatible with siRNA, sensitive to serum, or are capable of inducing cytotoxicity, which negatively affects gene expression studies.
High Throughput siRNA Screens
RNAi can be used as a research tool to study the function of single or many thousands of genes in cells. siRNA libraries composed of large numbers of siRNAs have been developed and are used to identify sets of genes functionally involved in a particular biological process or disease metabolism. Phenotypic high throughput siRNA screens are a powerful approach to study structure/function relationships of genes and to identify novels parts of signaling pathways. In fact, large-scale siRNA screening is now commonly used for functional genomics and drug target validation. High throughput applications require that reliable and reproducible transfections can be performed in 96-, 384-, or 1536-well plate format. Several transfection reagents are compatible for high throughput siRNA, shRNA, and miRNA transfection. A technique known as reverse transfection has been developed which allows for the plating of the cells and delivery of siRNA-reagent complexes on the same day thereby eliminating a source of high degrees of variability in cell culture and transfection efficiency.
Gene Silencing (RNAi) in Neuronal Cells
A number of studies have demonstrated the successful application of RNAi in primary cell cultures and non-neuronal cell lines; however the use of gene silencing in neuronal cell types has been notoriously difficult because of several technical and biological limitations. Neurons have been considered the most resistant to RNAi. Methods to consider when performing nucleic acid delivery experiments with neuronal cultures are: electroporation, microinjection, viral-based methods, and certain chemical transfection reagents. Although physical delivery methods appear to be efficient for siRNA-induced studies where transient modification of gene expression is sufficient, these methods do not appear to be adequate for behavioral studies where long term alteration of gene expression may be necessary. The viral-based siRNA expression systems appear to be most applicable for both Central Nervous System (CNS) gene therapy and basic neurobiological studies, while adeno-associated virus (AAV) delivery systems enable long-term, stable gene expression with relatively little cytotoxicity. Lentiviral vectors have been reportedly used successfully for the delivery of short hairpin RNA (shRNA), a precursor of siRNA, into primary neurons to induce RNAi. The advantage of lentiviral system is the ability to transfect non-dividing cells.
The capability of siRNAs to mediate post-transcriptional gene silencing in mammalian cells and tissues, as well as its successful use to prevent expression of target mRNA has recently led to the development of a new methodology for novel drug discovery. Several pharmaceutical and biotechnology companies are currently investigating the possible use of synthetic siRNA for inducing RNAi in vivo, its use in animal models, and in RNAi-based therapeutics. Small RNA molecules, such as siRNA, shRNA, and microRNA have been regarded as potential therapeutic agents to target multiple misregulated cellular processes therefore it is theoretically possible that RNAi can be utilized to treat any disease associated with over expression of specific genes. In fact, there are many reports in the literature that address the potential therapeutic application of RNAi to specifically target genes involved in multiple diseases including various forms of cancer, Alzheimerĺs, and a number of inflammatory and virally-associated diseases. However, a number of major difficulties associated with inefficient delivery of functional RNA molecules into cells and the reduced biostability of unmodified RNA must be overcome. Therefore, our research is focused on the development of efficient in vivo reagents and RNAi delivery technologies. Efficient and organ-specific delivery of synthetic oligonucleotide molecules is currently a key limiting step to enable siRNA- and microRNA-based therapeutic approaches.
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