Several techniques are available to alter gene expression in stem cells. These techniques include cell transfection via electroporation, lipid-mediated delivery or biolistics particle delivery as well as cell transduction though viral mediated gene delivery. These methods enable somatic cells to be reprogrammed into induced pluripotent stem cells and allow stem cells to be engineered for multiple therapeutic purposes. In addition to enabling gene expression, transfection and transduction techniques can be used to knock down gene expression levels via the RNA interference (RNAi) pathway.
In general, stem cells are considered difficult to transfect. The method used for transfection of stem cells varies depending on the cell type, the molecule being delivered and the downstream application. The most widely used methods are electroporation and lipid-mediated delivery. Biolistics is also a technique that may be useful for transfer of materials into stem cells. No single transfection method will work for all stem cells, and even within a lab, the method of choice may vary. These challenges have led many researchers to adopt viral-transduction methods for gene delivery.
Related Topics: Stem Cell Research, Isolation and Maintenance of Stem Cells, Differentiation of Stem Cells, and Analysis of Stem Cells.
Electroporation is a physical method in which an electrical shock is applied to the outer membrane to temporarily disrupt the lipid bilayer, allowing molecules to enter the cell. Since this method introduces only the molecule of interest, it is the method of choice for many investigators working in the area of gene therapy. Electroporation should be optimized depending on the cell type used. For example, the optimal conditions identified for electroporating human embryonic stem cells (hESCs), which resulted in 10–30% of cells showing strong, transient green fluorescent protein (GFP) expression, were a voltage of 250 V, a capacitance of 200 µF, a resistance of 1000 Ω, and an exponential decay waveform. On the other hand, mouse embryonic stem cells (mESCs) required a voltage of 220 V, a capacitance of 950 μF, and a resistance of 1000 Ω (Zsigmond 2009).
Bio-Rad has an extensive array of optimized electroprotocols for delivery of materials into a variety of cell types and cell lines. More protocols for electroporation can be found at www.bio-rad.com/transfectionprotocols.
Gene Pulser MXcell™ electroporation system and electroporation buffer for transfection.
Mouse ES cells grown in the presence of STO feeders (ES+) were electroporated using the Gene Pulser Xcell electroporation system with an exponential waveform at 240 V and 75 µF.
Lipid-mediated delivery is generally performed using commercially available lipid reagents. The lipid is mixed with the molecule being transfected and added directly to the plated cells. The lipid-encapsulated molecule is thought to move into the cell via endocytosis. Each lipid reagent must be optimized for the type of cell used. Efficiencies and toxicity of the lipids vary depending upon the reagent and the cell type. The disadvantage of this method is the introduction of an exogenous substance (the lipid) into the cell, which may alter biochemical pathways affecting pluripotency of the cell.
Biolistics particle delivery uses a high pressure burst of helium to "shoot" the molecules of interest into cells. The molecules of interest are first coated onto micron-sized beads. The method entails three steps: coating microparticles with DNA, 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. Cells penetrated by the microparticles are likely to become transfected.
Like the other techniques, a certain amount of optimization is required for this method. This includes optimizing the density of cells plated, the amount of microparticles used in each blast, 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. As ES cells differentiate into embryoid bodies, this technique could be employed. Bio-Rad's Helios® gene gun and PDS-1000 / He™ and Hepta™ Systems use a helium pulse to accelerate gold or tungsten particles coated with DNA directly into target cells.
More information on this technique and products can be found at www.bio-rad.com/biolistics/. Specific protocols for transfecting neuronal cells can be found at www.natureprotocols.com/2009/07/09/transfection_of_rat_or_mouse_n.php.
Viral-mediated delivery was the first method used, and the most efficient technique, to introduce genes into a somatic cell to reprogram it to become an induced pluripotent stem (iPS) cell (Takahashi and Yamanaka 2006, Okita et al. 2007, Takahashi et al. 2007, Wernig et al. 2007). This section will focus on the methodology used to produce the iPS cells; these are commonly used techniques that can be used to introduce genetic material into many cell types.
Retroviruses such as lentivirus have been the most commonly used for reprogramming; however, the lack of control over the gene integration site has led to the development of other viral vectors. A sendai virus, which carries out replication in the cytoplasm, has also been used to reprogram cells to the iPS cell state (Fusaki et al. 2009). Additionally, a polycistronic virus that reduces the integration events while still producing multiple proteins has been engineered, reducing the number of viruses necessary for inducing pluripotency (Carey et al. 2009).
Although viral-mediated gene delivery is highly effective, the production of the virus itself is time consuming. Lentiviral production is accomplished using three separate constructs — a transfer vector, a packaging plasmid, and an "envelope" plasmid. The virus is produced by first cloning the DNA of interest into the transfer vector. The appropriate clone is selected and transfected with the packaging vector and "envelope" plasmid into 293T cells overnight to propagate the virus. Supernatant from the cells containing the amplified virus is then titered to determine the concentration. Virus prepared in this manner can generally be stored for several weeks at 4°C.
More information on viral production can be found at the following sites: http://biology.kenyon.edu/slonc/gene-web/Lentiviral/Lentivi2.html www.broadinstitute.org/genome_bio/trc/protocols/trcLentiVirusProd.pdf http://web.mit.edu/jacks-lab/protocols/lentiviralproduction.htm
Carey BW et al. (2009). Reprogramming of murine and human somatic cells using a single polycistronic vector. Proc Natl Acad Sci USA 106, 157–162. PMID: 19109433
Fusaki N et al. (2009). Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proc Jpn Acad Ser, B Phys Biol Sci 85, 348–362. PMID: 19838014
Okita K et al. (2007). Generation of germline-competent induced pluripotent stem cells. Nature 448, 313–317. PMID: 17554338
Takahashi K and Yamanaka S (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676. PMID: 16904174
Takahashi K et al. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872. PMID: 18035408
Wernig M et al. (2007). In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448, 318–324. PMID: 17554336
Zsigmond E (2009). Transfection of mouse and human embryonic stem cells by electroporation using the Gene Pulser MXcell™ system. Bio-Rad Bulletin 5904.
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