Several techniques are available to modify gene expression in stem cells, including transfection of nucleic acids into cells. Transfection methods comprise electroporation, lipid-mediated delivery, and biolistic particle delivery, as well as cell transduction though viral-mediated gene delivery.
Transfection is used to reprogram somatic cells to induced pluripotent stem cells (iPSCs), and to engineer stem cells for therapeutic or research purposes. In addition to facilitating gene expression, transfection and transduction techniques can be used to downregulate gene expression levels via RNA interference (RNAi).
Stem cells can be difficult to transfect. The methods used for stem cell transfection vary with cell type, species, the molecule being delivered, and the intended downstream application. Electroporation and lipid-mediated delivery have been the most common methods for the transfection of stem cells. Biolistic particle delivery is a cell-type independent technique that can be used to transfer genetic materials into stem cells. The challenges of chemical and physical transfection methods have led many researchers to adopt viral-transduction methods for gene delivery in stem cells.
Electroporation temporarily disrupts the plasma membrane lipid bilayer with an electric shock, allowing molecules to enter the cell. Electroporation protocols are optimized for various cell types. Bio-Rad has determined the optimal electroporation protocols for delivery of materials for most common cell types and cell lines. The Transfection Protocol Library contains standard and customized user-submitted protocols for a wide variety of cells (select cell type and protocol type from the dropdown menus).
Lipid-mediated transfection uses cationic lipids to deliver DNA and RNA into cells. Transfectin™ Lipid Reagent, a mixture of cationic lipids and a co-lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) has been used to transfect stem cells such as neural stem cells (Sun et al. 2010, Lang et al. 2012).
In lipid-mediated transfection, lipid is mixed with nucleic acids and then added directly to the cells. Lipid-encapsulated molecules are thought to enter the cell via endocytosis. Transfection efficiency varies depending on the reagent and the cell type. Lipid toxicity can sometimes be a problem when transfecting cells. Transfectin Lipid Reagent has low toxicity and is effective in stem cell transfection protocols.
Biolistic particle delivery is the injection of small compounds, proteins, or nucleic acids into cells by coating them onto inert micron-sized particles, which are shot directly into cells as microprojectiles using a high-pressure burst of helium.
The Helios® Gene Gun and PDS-1000 / He™ and Hepta™ Systems use a helium pulse to accelerate gold or tungsten particles coated with DNA, RNA, or protein into target cells. Biolistic delivery entails three steps: coating microparticles with DNA (or other transfectant), drying them onto a macrocarrier disk, and propelling them into the target cells. The macrocarrier disk is accelerated with high-pressure helium into a stopping screen, which frees the microprojectiles to bombard the cells.
Like the other techniques, optimization is required for cell type and source. Optimization includes the density of cells plated, the quantity of microparticles used, the pressure used to deliver the particles, and the amount of vacuum applied. A major advantage of the biolistic approach is the ability of the particles to be carried through many layers of cells, for example, effectively transfecting 3D cultures or embryoid bodies.
Other advantages of biolistic particle delivery systems include the ability to codeliver more than one plasmid or other nucleic acid, their effectiveness with all cell types, the fact that no carrier is needed, the low levels of transforming DNA required, and their additional use for the delivery of proteins and RNA, including modified RNAs such as miRNA mimics.
Viral gene delivery, or transduction, was the first method used to introduce genes into somatic cells to reprogram them to become iPSCs (Takahashi and Yamanaka 2006, Okita et al. 2007, Takahashi et al. 2007, Wernig et al. 2007).
Gamma-retroviruses have commonly been used for cellular reprogramming; however, the lack of control over the gene integration site has led to the development of protocols using different viral vectors. A polycistronic doxycycline-inducible lentivirus that reduces the frequency of integration events while still producing multiple proteins has been engineered (Carey et al. 2009), lowering the risk of deleterious proviral insertion. Although viral-mediated gene delivery can be effective, the production of the virus itself is time consuming. Lentivirus production is accomplished using three separate constructs — a transfer vector, a packaging plasmid, and an "envelope" plasmid. The addition of enzyme inhibitors, micro RNA (miRNA), and miRNA mimics have been found to increase lentiviral transduction efficiency (Anokye-Danso et al. 2012). Sendai virus, an RNA virus (Fusaki et al. 2009, Seki et al. 2012), and adenovirus (Stadfield et al. 2008), neither of which integrate into host genomes, have also been used to reprogram somatic cells.
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