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The Right Tool for the Job

Tue, 07/05/2011 - 11:07am
Jeffrey M. Perkel

Hard-to-transfect cells can create difficulties when selecting a method for nucleic acid delivery.

At some point in most every molecular biology project, cells will need to be transfected. There’s simply no better way to know what a gene or putative regulatory element is doing than to insert it into cells and see what happens.

Researchers generally have three choices for nucleic acid delivery: transfection reagents, electroporation, and viruses. (A fourth option, biolistics, is used mostly for agricultural work). Whether its plasmids, bacterial artificial chromosomes, or short RNAs, for many standard cell lines any option will do; the choice is a matter of finding the easiest, most cost-effective, and most appropriate tool for the desired application. For certain hard-to-transfect cells, however, such as suspension and primary cells, the answer is less obvious. Fortunately, reagent and device manufacturers continue to develop new tools for the scientific community.

Transfection Reagents
The fundamental problem with transfection is that cells are (not surprisingly) very different. For instance, says Patrick Erbacher, CSO at Polyplus Transfection, the endocytic pathway typically is “well developed” in adherent cells, but less so in suspension cells. Primary cells and stem cell-derived cell types typically divide slowly (or not at all) in culture, says Laura Juckem, R&D Group Leader at Mirus Bio, whereas immortalized cancer cell lines divide relatively rapidly. These variables can strongly influence the efficiency with which a given cell type will take up and express exogenous nucleic acids.

“Cells are like people: We are all very different,” says Michelle Collins, senior global product manager in the Gene Expression Division at Bio-Rad Laboratories, a company that offers a range of nucleic acid delivery tools, including the TransFectin and siLentFect lipid reagents.

 Mechanism of DNA and siRNA transfection in Eukaryotic cells 
click to enlarge
Mechanism of DNA and siRNA transfection in Eukaryotic cells. (Source: Polyplus-transfection SA)

Nucleic acids also are very different. Plasmid DNAs have different physical characteristics than siRNAs or mRNAs, for instance, and must make their way to the nucleus to be functional, as opposed to RNAs, which work in the cytoplasm.

The upshot is that what works for one cell type or class of nucleic acid, may not work for another.

“There is no magic bullet in this business,” says Paulina Leung-Lee, transfection product manager at EMD Millipore. “You have to tailor the delivery reagent based on what you are trying to deliver, and where you are trying to deliver it.”

EMD Millipore’s portfolio includes the GeneJuice transfection reagent (for plasmid DNA delivery to most established cells), RiboJuice siRNA reagent, and Insect GeneJuice, a GeneJuice variant specifically intended for insect cells. These reagents vary from lipid-based to novel polymer-based formulations. For hard-to-transfect cells, the company offers NanoJuice, which is dendrimeric.

The key, says Adrian Vilalta, Molecular Biology R&D team Lead at EMD Millipore, is to balance transfection efficiency, toxicity, and scalability. EMD Millipore’s NovaCHOice Transfection Reagent, slated to launch in April and specifically designed for scalable biomanufacturing applications using CHO cells, “hits all three points very nicely,” says Vilalta, offering 32% higher expression on day 1 post-transfection than other methods, with low toxicity and the ability to scale easily from 50 mL to 5L.

EMD Millipore is not alone in offering multiple formulations depending on the intended cell type or molecule to be delivered. Life Technologies’ Lipofectamine 2000 reagent is intended for in vitro delivery of plasmid DNA and small RNAs, whereas its Lipofectamine LTX targets plasmid DNA in difficult-to-transfect cells and Lipofectamine RNAiMAX addresses small RNAs.

Similarly, Roche Applied Sciences offers various formulations of its X-tremeGENE reagent for generic and hard-to-transfect cells, as well as siRNA, as does QIAGEN with its Attractene, Effectene, and HiPerFect reagents. Mirus Bio supplies its TransIT transfection reagent in cell-type-specific formulations for HEK293, HeLa, and Jurkat T cells, among other targets.

NovaCHOice Transfection Kit
The NovaCHOice Transfection Kit is quality controlled against suspension-adapted Chinese hamster ovary (CHO) cells. The kit is formulated to offer highly efficient DNA delivery with minimal cell death to maximize recombinant protein expression. (Source: EMD Millipore)

Though most of these reagents work more or less the same way—mix nucleic acid with reagent, form complexes, add them to cells, and test 24 to 72 hours later—different reagents do employ different mechanisms. Perhaps the most common formulation is the cationic lipid—an amphiphilic molecule that encapsulates the nucleic acid in a micelle and interacts with the cell surface via electrostatic interactions.

There are other approaches. EMD Millipore’s NanoJuice is a dendrimer, while Polyplus Transfection’s reagents employ cationic polymers such as polyethylenimine (PEI). As Erbacher explains, transfection reagents mainly enter into cells via endocytosis. In the case of cationic lipids, subsequent release of nucleic acids from the endosomes into the cytoplasm eventually occurs via a membrane fusion mechanism. Cationic polymers such as PEI also use endocytosis, Erbacher explains; but they act as “proton sponges” in the endosome, absorbing the compartment’s acidity and causing it to swell and rupture.

Of course, that’s in vitro; transfection in vivo is another problem entirely. “The difference between in vitro and in vivo is that in vitro, you can put [the transfection complexes] directly on cells; in vivo you need to be resistant in blood and avoid clearance,” says Xavier de Mollerat du Jeu, senior staff scientist at Life Technologies. The company’s Invivofectamine 2.0 reagent offers highly efficient delivery of siRNA to the liver via tail vein injection.

Electroporation
When it comes to nucleic acid delivery, researchers typically start with the easiest options and work their way down from there. As a result, says Gerhard Muster, product manager at Lonza, the usual progression is reagents, electroporation, and viral transduction. “Reagents are cheap but not applicable to hard-to-transfect cells, whereas viral transduction needs a hell of a lot of work to do.”

In electroporation, Collins explains, an applied electrical pulse causes cellular pores to open, enabling foreign DNA to enter the cell. Once the pulse is removed, the pores close, locking the material inside the cell. The benefit of this approach, Muster says, is that it is highly efficient; the disadvantage is, it can be highly toxic.

The granddaddy of electroporators is Bio-Rad’s Gene Pulser system, available in both single cuvette- (Gene Pulser Xcell) and plate- and cuvette-based (Gene Pulser MXcell) configurations. Gene Pulser is an “open” system: the user has complete control over the strength and characteristics of the electrical pulses used to deliver the nucleic acids into cells. So is Life Technologies’ Neon system, which swaps traditional electroporation cuvettes for gold-plated pipette tips. By contrast, Lonza’s Nucleofector systems are “closed”—users cannot tweak electrical parameters directly, but rather optimize conditions using different combinations of preset protocols and solutions. “The aim is to give customers a tool that leads to results, not playing around with individual parameters for months,” Muster says.

With both the Gene Pulser and Neon, the cells are transfected in solution, meaning adherent cells must be released from their substrates. Though it adds an extra step to experimental protocols, detaching cells has not historically presented a major problem; “People have been detaching cells for various experiments for decades,” Collins says. For some very fragile primary cells, and for specialized cells like neurons, however, detachment could influence cell viability and physiology.

Starting this fall, though, researchers will have another option. Lonza’s 4D-Nucleofector is a modular system comprising a core controller module and an “X module” capable of transfecting from 104 to 107 suspension cells in cuvettes or 16-well strips. The system employs conductive polymer electrodes rather than metal for lower toxicity. A new “Y” module, currently in beta testing, will enable transfection of adherent cells in 24-well plates directly, without first detaching them.

Viral Reagent
A third approach to nucleic acid delivery is viral transduction, commonly achieved using lentiviral systems. Lentiviral reagents are available from Life Technologies, Thermo Fisher Scientific, and Sigma-Aldrich, among other companies.

According to Supriya Shivakumar, global commercial marketing manager for functional genomics at Sigma-Aldrich, lentiviral systems offer two distinct advantages: Lentiviruses infect non-dividing cells, and can generate stable transfectants. “This [approach] allows you to deliver nucleic acid in such a way that it is integrated into the genome,” Shivakumar explains.

The disadvantage is that somebody has to make the infective viruses first. In many cases, somebody already has.

One relatively recent example: Sigma’s line of MISSION 3’UTR Lenti GoClone viruses. Every human 3’UTR is represented, controlling the expression of a luciferase reporter. Simply select the human gene of interest, and Sigma-Aldrich will supply a replication-incompetent, ready-to-use viral stock. Following transduction, researchers can determine which (if any) microRNAs regulate that gene by introducing the small RNAs and monitoring their impact on luciferase expression.

Considerations
When selecting a transfection strategy, it’s best to plan ahead, says Collins. “You need a realistic roadmap of where you’re going,” she says. What cell lines and types will you be using, and what kind of nucleic acids? Is transient transfection sufficient, or do you need stable transfectants?

Mirus Bio’s Juckem recommends running a direct head-to-head comparison using different approaches on the same day. “That gives the most accurate picture,” she says. “When comparing results from different days things can get confusing.”

Finally, check the literature: have others successfully transfected these cells? Online cell line databases chronicle user experiences with different reagents, protocols, and cell types. Says Life Technologies’ de Mollerat du Jeu of the company’s Protocol Exchange, it’s “a great tool to avoid redundancy and all these painful steps [of protocol optimization]. Otherwise, it’s just a waste of time for everybody.”

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