May 15, 2006 (Vol. 26, No. 10)
Kathy Liszewski
Coaxing Cells to Efficiently Express their Protein Payload
Delivering DNA into cells is a rapidly developing area of biotechnology and gene therapy. Additionally, it is a powerful research tool for deciphering gene structure, function, and regulation. Nonviral methods for gene delivery center on the use of chemical formulations that complex DNA or technologies that provide a gentle electrical shock to pry open cellular pores for DNA entry.
Obstacles to overcome include keeping the cells viable by reducing toxicity and severity of chemical or electrical exposure and of course, enhancing efficiency and reproducibility of DNA uptake to drive high level expression.
A number of companies have offerings for nonviral methods of DNA delivery. Scientists transfect a variety of cell lines, a process often requiring individual optimization. Aiming to streamline the process, ATCC (www.atcc.org) and nonviral gene transfer specialists at amaxa (www.amaxa.com) joined forces. Using its Nucleofector technology on cell lines supplied by ATCC, amaxa is creating and optimizing transfection protocols.
For optimal results with any assay, ATCC has traditionally recommended using new, low-passaged cell lines, explains Peyton Hughes, business unit director cell biology at ATCC. Likewise, amaxa knew that its transfection customers experienced better results with cultures directly purchased from ATCC and often recommended tossing out the over-passaged lines or those obtained from a colleague.
Around the time that amaxa started suggesting that researchers go back to genuine ATCC lines, ATCC identified amaxa as offering a new solution for the efficient transfer of nucleic acids using Nucleofector technology. So, our two companies started a collaboration to optimize protocols and cross-reference them with the cell lines on our Websites.
The Nucleofector technology, marketed by amaxa, uses a unique combination of electrical parameters and proprietary solutions that infuse DNA or RNA directly into the cell nucleus. While other methods require dividing cells for transfection, nucleofection works on both dividing and nondividing cells. The technology provides rapid expression (2𔃂 hours) with transfection efficiencies of 50󈟆%, according to the company.
Importance of Optimization
Bio-Rad Laboratories (www.biorad.com) offers both transfection reagents and electroporation technology. For example, TransFectin is composed of a cationic lipid and a colipid called DOPE (1,2-dioleoylphosphatidylethanolamine).
The principle is fairly straightforward. The cationic lipids interact with DNA or RNA in aqueous solutions to form positively charged micelles or liposomes. These lipid-nucleic acid complexes fuse with the membrane, become internalized, and subsequently expressed.
We have optimized TransFectin for a variety of commonly used cell lines, such as CHO, NIH-3T3, A549, 293, and even challenging primary cell lines, says Steve Kulisch, marketing manager. Remarkable transfection efficiencies can be achieved by adjusting parameters, such as cell density, ratios of TransFectin and DNA, and incubation time.
Kulisch also notes that Bio-Rad gave a face-lift and added functionality to the Gene Pulser with the release of the Gene Pulser Xcell electroporation system. When first released in 1986, the Gene Pulser system was used primarily for bacterial transformation. But now we have enhanced its capabilities for mammalian cells.
Innovative Hypoosmolar Buffers
Eppendorf (www.eppendorfna.com) offers its Multiporator for the transfection of animal and human cell lines, embryonic stem cells, primary cells, plant protoplasts, bacteria, and yeasts.
This model is compact, portable, and easy to operate, according to Joel Lopez, product manager. Our clients not only get high transfection efficiencies but also great reproducibility. We have taken a careful look at plastics, electrodes, and buffers. All of these can affect outcome.
We offer reagents and cuvettes that are optimized for our own system. This is an advantage. Most companies offer only the instrument and maybe cuvettes. Our buffers are specially developed to feature low conductivity and low osmolarity. The low conductivity boosts cell survival, allowing use of reduced voltage. Additionally, the low osmolarity of the buffer enhances pore formation.
Lopez suggests that the design of the instrument offers another advantage in that the Multiporator uses soft pulse technology. This provides for exponential decay of pulses rather than the traditional square wave pulsing. Exponential decay is like a gentle slope that dissipates over time. Overall, this is a much more cell friendly approach.
Invitrogen (www.invitrogen.com) offers transfection reagents based on its cationic lipid formulations. The company’s flagship product, Lipofectamine 2000, provides an easy protocol that works efficiently for plasmids and RNAi transfections. Customers like this product, because it works reliably on a broad range of cell lines, explains Henry Chiou, Ph.D., research manager for transfection.
We also offer products with different chemistries and formulations for specialized applications. For example, Oligofectamine is used for oligonucleotide transfections.
Invitrogen is launching several new products this year, according to Dr. Chiou. These are next-generation reagents. A limitation of current technologies is the requirement for optimizing cell densities and reagent dose. We’ve developed new reagents that expand the transfection sweet spot for more applications. These products expand the optimal working range for both reagent dose and cell density. For example, Lipofectamine LTX is designed for plasmid, shRNA, and miRNA vectors. Lipofectamine RNAiMAX is designed for short RNAi duplexes. These new reagents provide high transfection efficiency and lower cytotoxicity.
We are also introducing the Freestyle MAX Expression System for rapid protein production in suspension CHO and HEK293 cells. There is huge interest in transient transfection in suspension culture to produce large amounts of protein. This system provides all components for this application to ensure optimal compatibility, including serum-free medium, transfection reagent, and CHO and HEK293 cells, adapted to serum-free suspension culture.
High-Throughput Transfections
Mirus Bio (www.mirusbio.com) has been providing transfection reagents for more than a decade. Its TransIT technology is designed for a broad spectrum of nucleic acid delivery applications, including plasmid and siRNA transfections. For example, the TransIT-LT1 reagent is a polyamine-based reagent that enables high-efficiency, low-toxicity plasmid delivery into a variety of cell types using serum-containing media. Additionally, the reagent is adaptable for use in a 96-well plate format for high-throughput applications.
The cell line specific TransIT reagents are optimized for particular cell lines, including difficult-to-transfect cells, such as Jurkat, notes Claire Ruzicka, Ph.D., director of marketing. The TransIT-293 reagent offers an alternative method to calcium phosphate for transfecting multiple plasmids for viral production in 293 cells.
For clinical applications, Mirus has developed the Pathway IV technology for the intravenous delivery of plasmid DNA to targeted limb muscles. After delivery to muscle tissue, the DNA expresses therapeutic proteins that are active locally or systemically.
Applications for this technology are muscle-specific illnesses, such as muscular dystrophy and multiple sclerosis, says Dr. Ruzicka. This method overcomes typical delivery hurdles because it relies on a simple and elegant means of restricting blood flow to increase permeability of blood vessel cell walls in the area of injection. The DNA quickly passes through the cell walls and into adjoining muscle tissue.
If a spoonful of sugar helps the medicine go down, a dab of nontoxic cellular protein may assist transfection efficiency. EMD Biosciences (www.emdbiosciences.com), under its Novagen brand, provides GeneJuice transfection reagent for transfection of DNA into mammalian cells.
Adding Protein to Enhance Efficiency
Whereas many available transfection reagents are based on cationic lipid formulations, GeneJuice consists of a nontoxic cellular protein and a novel polyamine, says Craig Draper, Ph.D., product manager for analysis and array products at Novagen.
The idea is to provide a more gentle and supportive environment for the cellular disruption needed for transfection, according to Dr. Draper. The exact mechanism of how transfection actually works is not known. Basically, you coax cells into opening up enough for the passage of DNA into them, yet not so much as to harm the cell.
Our approach uses a unique chemistry that offers a number of advantages over lipid-based transfections, such as high-efficiency transfer for both stable and transient transfections, minimal cellular toxicity, compatibility with serum, and a simple protocol that does not require media changes.
The technology can be used on a variety of cells and for high-throughput transfections in a multiwell plate format, adds Dr. Draper. The company also offers RiboJuice siRNA transfection reagent that delivers siRNA into a number of mammalian cell lines for targeted gene suppression, Insect GeneJuice for transfection of insect cells, and ProteoJuice protein transfection reagent for the introduction of intact functional protein into mammalian cells with minimal toxicity and broad cell specificity.
Activated Dendrimers
Qiagen (www.qiagen.com) provides a transfection technology, based upon activated-dendrimers chemistry. Dendrimers are highly branched, three dimensional, multifunctionalized polymers.
Historically, scientists used liposomes for DNA delivery, comments Constanze Kindler, Ph.D., senior global product manager transfection. We were the first company to offer something completely different. The idea of the dendrimer is that it possesses a defined spherical architecture. It has branches radiating from a central core that terminates at charged amino groups. So, the PolyFect transfection reagent can condense DNA into small, compact structures that are taken up more easily by eukaryotic cells.
The company offers optimized protocols for a number of cell lines, including COS-7, NIH/3T3, HeLa, 293, and CHO cells. There are a number of advantages to this technology, according to Dr. Kindler. It’s fast and easy, economical, highly efficient, and can be used in the presence of serum.
Qiagen also offers Effectene, which is formulated especially for primary cells and other cell lines needing gentle conditions for transfection. In this case, the reagent is used in conjunction with the company’s Enhancer and a DNA-condensation buffer to achieve high transfection efficiencies.
One of the advantages of Effectene is that it spontaneously forms micelles that show no size variations as seen in typical preformulated liposome reagents, Dr. Kindler notes. So, this is a unique feature that helps ensure excellent reproducibility of transfection complex formations.
Qiagen also offers reagents for siRNA delivery, including HyPerFect and RNAiFect.
Minimizing Optimization
Roche Applied Science (www.roche-applied-science.com) markets FuGENE 6 transfection reagent. This lipid formulation produces high levels of transfection with minimal cytotoxicity for many eukaryotic cell lines. An added benefit is the ability to perform transfections in the presence or absence of serum. Hence, only minimal optimization is required, according to Roche.
The company suggests that FuGENE 6 typically yields >90% cell viability following transfection (as measured by COS-1 cells). To enhance success, they recommend using highly pure nucleic acids, cells that are in growth phase, and optimizing the ratio of DNA to transfection reagent.
Roche also markets X-tremeGENE Q2 that is designed and optimized for difficult-to-transfect cell lines, K-562, and Jurkat cells.
Reducing Cytotoxicity
Keeping cells happy during the process of transfection can spell the difference between success and failure.
A lot of transfections can be hit or miss due to cell toxicity, says Ben Price, product manger cell biology at Stratagene (www.statagene.com). We offer GeneJammer reagent, which has demonstrated exceptionally low cytotoxicity and enhances the number of viable cells. This product is based on a formulation of novel polyamines that can be used for both transient and stable transfections for a wide variety of cells lines. It’s fast and easy to use and doesn’t require media changes. It’s very efficient even in difficult to transfect cells lines. Another advantage is that it works equally well with or without serum, antibiotics, growth factors, and other supplements.
But no one way is universal, according to Price. Stratagene also offers LipoTAXI. Some similar lipid-based technologies are toxic, but LipoTAXI is formulated to be gentler on cells and typically demonstrates low cellular toxicity. It also consistently yields high transfection efficiency, typically a 10-fold increase over methods such as calcium phosphate/DEAE mediated transfections. We’ve used this reagent in more than 30 cell lines and primary cultures.
Thermo Electron (www.thermo.com) offers its Cellject Duo. This electroporation instrument has been on the market for several years and is an established technology, suitable for most cell types, says Mike Campisano, product manager. You can program by cell type or parameter values. The user can easily select standard parameters for the type of cells used. We have also created a Protocol Passport with specific conditions for a variety of cell lines. For scientists who already have developed their own protocols, the instrument comes with external program cards so users don’t have to re-enter their programs.
Another advantage of the Cellject Duo is its remote electroporation chamber. It’s small enough to easily be used in a laminar flow hood. The size makes it easier to keep essentials like pipettes and ice right at hand for fast processing and improved cell survival, notes Campisano.
Thermo also offers an optimized buffer, Optibuffer, which provides additional salts and critical molecules to help in the regeneration process, following the destabilization produced by the electrical charge to cells. Additionally, it markets universal cuvettes for smaller sample sizes using a V design in the base.
