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GEN UPDATES in biotechnology: Transfection

In Vitro Nucleic Acid Transfection Methods
Patricia F. Dimond, Ph.D.

The introduction of exogenous nucleic acids into eukaryotic cells through transfection has enabled analyses of gene function, expression, regulation, and mutation, which has advanced basic cellular research, drug target identification, and validation. It has also made the production of human therapeutic proteins feasible.

Transfection technologies include chemical and lipid-mediated reagents or physical introduction through microinjection and electroporation. Methods combining physical carriers like nanoparticles with chemical or lipid reagents are also becoming available.

Access to a plethora of commercially available transfection tools has facilitated, but to some extent commoditized, transfection and obscured the reality that there are no one-size-fits-all experimental situations and guarantees of success.

The choice of a given transfection reagent or technology can significantly impact experimental outcomes and the interpretation of results. Relatively novel nucleic acid molecules such as siRNA, for example, pose particular challenges for delivery methods. The ability to specifically silence target genes at optimally low concentrations and without producing off-target effects depends on the efficiency of siRNA delivery as well as the siRNA sequence itself.

Many cell types (e.g., primary cells, stem cells, and those grown in suspension culture) remain challenging transfection targets. The following discussion addresses some of the basic considerations affecting the choice of a transfection method, currently used techniques and their limitations, and newer approaches aimed at overcoming these. While methods including electroporation or particle guns may be used for transfection, this review focuses only on chemical approaches.

Currently Used Chemical Transfection Methods

The goal of a chemical transfection reagent, as with all transfection technologies, is to introduce foreign negatively charged molecules (primarily nucleic acids) into eukaryotic cells that have negatively charged membranes for expression of a given gene of interest. Certain chemicals and non-lipid cationic polymers, as well as cationic lipids, neutralize or impart an over-all net positive charge to nucleic acids, allowing them to cross membranes and may, to some extent, protect them from degradation once inside the cell as they make their way to the nucleus.

Non-lipid chemical reagents used for transfection include calcium phosphate, DEAE-dextran, and cationic non-lipid polymers. In general, these chemical agents become complexed with nucleic acids, giving them a neutral or net-positive charge so that they can cross cell membranes.

For example, plasmid DNA and calcium phosphate form a co-precipitate, which settles onto the surfaces of adherent cells and is then endocytosed. These reagents were some of the first technologies developed for transfection and continue to be used today.

While reagents such as calcium phosphate have well-established methods and a relatively low cost they can be limited in breadth of application and functionality across cell lines.

Lipid-based Reagents

Lipid-based reagents, based on cationic lipid polymers, include liposomes and linear or branched cationic polymers. These too form complexes with nucleic acids and facilitate their transport across cell membranes via endocytosis. Liposomes, spherical structures consisting of single or multiple concentric bilayers, result from the spontaneous assembly of amphiphatic molecules in aqueous environments. Liposomes are composed of synthetic versions of the cationic phospholipids that comprise cellular membranes. Positively charged moieties on the phospholipids associate with the negatively charged phosphate groups of nucleic acids thereby neutralizing them. The net positive charge of the resulting complex reduces electrostatic attraction of the nucleic acid to the negatively charged cell membrane.

The success of lipisomal-based transfection reagents depends on lipid formulation, charge ratio, particle size, method of preparation, and the cell type. Generally, cationic lipids perform well in vitro; liposomal delivery offers relatively higher efficiency of gene transfer, infectivity of a larger range of nucleic acid sizes to more cell types than chemical methods and, for both, transient and stable integration. However, because of the variables inherent with liposomalbased reagents, they tend to be specialized to particular cell types and nucleic acids.

Cationic Lipid Transfection Agents-Non-liposomal

Since Felgner described 5 to 100 times greater transfection efficacy than chemical methods using synthetic cationic lipids, many variations on these compounds have been developed. These compounds have reportedly achieved more efficient transfection in cell lines for which liposomal delivery has been relatively unsuccessful.

Cationic lipids may be mixed with neutral lipids such as Ldioeleoyl phosphatidylethanolamine (DOPE) to facilitate cross-membrane transfer and intracellular nucleic acid release. While the cationic portion of the lipid head group associates with the negative charges on nucleic acids, thus allowing compaction of the nucleic acid, the more neutral portion of the molecule is thought to facilitate fusion of the nucleic acid-polymer complexes with the cell membrane and encourage their release from endosomes intracellularly.

Transfection reagents based on this technology are broadly available and effective. But they can be relatively toxic to the cells being transfected depending upon experimental conditions and may impart off-target effects.

Synthetic Non-lipid Polymers

Synthetic non-lipid cationic polymers, in linear or branched conformations, can condense DNA into relatively smaller particles, which are endocytosed at the cell membrane. These reagents include commercial products and polyethyleneimine and polyamidoamine, and can include cationic proteins like polylysine, protamine, and histones.

Dendrimers are highly branched spherical molecules consisting of non-lipid polymers such as PAMAM. Positively charged groups on activated dendrimers bind DNA, thereby allowing formation of complexes (dendriplexes), compact structures then bind to cell membranes and are transported into the cell by nonspecific endocytosis.

Transfection reagents based on these technologies have been shown to have broad applicability across many cell eukaryotic cell lines and types. They impart high transfection efficiencies, produce high levels of protein expression, and, when experimental conditions are optimized, limit cytotoxicity and off-target effects.

Transfection Goals and Experimental Considerations

Broadly speaking, transfection of exogenous DNA into cells has two goals: expression of a desirable protein in useful amounts and cellular analysis. The choice of a transfection reagent or technology should be based on the experimental goals and whether those goals are best met through transient gene expression, or whether the experimental goals require stable, long-term gene expression.

In transient gene-expression experiments, exogenous DNA is introduced and expressed, but does not integrate into host chromosomes. Analysis for transcribed product is usually carried out within 12-72 hours post-transfection. The goal of stable, long-term gene transfection is to identify, isolate, and propagate cells that have integrated transfected DNA stably into their genomes.

Generation of stable transfectants requires the inclusion of a dominant selectable marker in the gene construct or cotransfection of the plasmid with a selectable marker. Stable transfection requires greater investment in time and effort to produce and isolate a stably transfected clone-one that be maintained and with limited variability in expression.

For any transient transfection experiment fundamental considerations include the choice of a cell line, growth medium, vector backbone, and the appropriate transfection reagents and technologies. All of these factors must be evaluated in the experimental design to achieve optimal results. The transfection reagent or technology has not always been considered a significant factor and this can limit the success of the experiment by reducing protein yields or producing results made suspect by off-target effects.

In most protein expression applications using eukaryotic cells, maximizing the yield of an appropriately modified, properly folded functional protein to allow economically feasible recovery and purification with minimal batch-to-batch variations is the primary goal. Transient transfection for the purpose of protein expression can be optimized with relatively little concern about the toxic effects upon cells if the end goal is maximal expression.

To increase protein-expression yields through use of largevolume bioreactors, many difficult-to-transfect suspension cell lines are employed. There are commercial transfection reagents that have been shown to produce high levels of protein expression through transient transfection of difficult to transfect suspension cell lines in serum-free media.

FuGENE® HD Transfection Reagent from Roche Applied Science (www.powerful-transfection.com) successfully combines efficient delivery of DNA into the cell lines commonly selected for protein expression, limits cytotoxicity, and dramatically increases yields of the target protein in media (serum-containing or -free).

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For cellular analysis experiments such as the determination of gene-expression effects on a particular regulatory pathway, transfection reagents should be selected to produce optimal results with minimal off-target impact on the cells. Investigators may be required, based on the study, to work with a specific cell type, which may be primary (neuronal, cardiac myocyte) as opposed to a cell line, which may be much easier to transfect. For primary cells, the culture medium usually requires serum or other growth factors. The gene delivery vector can't affect other gene functions, and the transfection reagent shouldn't interfere with normal gene expression, creating off-target effects or damage to the cell.

In the interest of achieving the best experimental result, as much care should be taken in choosing transfection methods and reagents as for other experimental parameters. Many commercially transfection reagents can provide high transfection efficiency with difficult to express cell lines but few are able to combine efficiency across a broad range of cell lines and types with minimal cytotoxicity and off-target effects.

"The issue of off-target effects generated from the transfection reagent used in an experiment has been overlooked in the past," says Jeffrey Emch, product manager with Roche Applied Science "We see this as one of the key advantages in application of FuGENE® HD Transfection Reagent."

Importance of the Cell in a Transfection Experiment-Cell Line Authentication and Cell Culture Maintenance

Starting out with authenticated, contamination-free cell lines is a critical component to ensure reliable and reproducible results with any experiment. It is also important not to keep cells too long in culture as this may lead to genetic drift and render the cells no longer reliable models of their original source material.

Therefore, to ensure optimal transfection results, the use of quality-tested cell lines is the first step. Employing good cell culture practices, which include monitoring growth, ensuring optimal seeding densities, and using defined passage numbers, is the second step to consistently achieve good transfection results.

ATCC, which tests cell lines starting with the depositor's original material and continuing through to every distribution lot, has a few suggestions to help researchers avoid the use of over-subcultured cells.

ATCC recommends monitoring cell lines routinely by examining morphology, establishing identity markers for genes of interest and/or developing criteria on growth rates, protein expression levels, or transfection efficiencies for use as baseline data. By comparing cell-line performance at various passage numbers to the baseline data and looking for changes or unexpected results, scientists can identify workable safe passage number ranges. Identifying and using defined passage numbers for specific cell lines will result in both higher transfection efficiencies and consistent protein expression.

According to James Greene, Ph.D., professor of cell biology and associate director of the Institute for Biomolecular Studies at The Catholic University of America, "Continuous subculturing selects for cell types that may differ from the original in subtle ways enough to materially impact results."

He suggests that such changes may be detected by comparative genomic hybridization (CHD), in which cells from different passage numbers of the same cell line can be compared with each other.

Summary

While current and future transient transfection reagents afford investigators a wide array of options, addressing all of the variables in line with the general purpose of the experiment is a challenge.

While a wide variety of transfection reagents and techniques are available to investigators, demands for greater efficiencies, wider applicability, and reagents enabling the execution of more complex experiments continue to foster inventive development efforts. These efforts will undoubtedly result in the continued evolution of increasingly sophisticated transfection reagents and technologies. July 2007

 

Effects of Passage Number on Cell-Line Transfection

An important factor in the success of any transfection experiment is the quality of the cells. As a key experimental component, the cell line can also be the greatest variable, affecting the reliability and reproducibility of results.

In transfection experiments, the passage number of the cell lines can affect not only transfection efficiency, but protein expression as well. The data shown in the figure demonstrate low and high-passage RAW 264.7 (ATCC® TIB-71™) cells transfect equally well, but protein expression is significantly reduced in the high-passage samples. RAW 264.7 cells were transfected with a plasmid for luciferase expression at passage number 5 (low passage) and 74 (high passage) using FuGENE® HD Transfection Reagent for comparative studies. Three volumes (4, 6, and 10 µL) of the same complex (5:2 ratio of reagent to DNA) were added to all cells.

Similar expression levels (top graph) were observed 24 hours post-transfection at either passage number. However, luciferase expression dropped off significantly 48 hours posttransfection in the high-passage cells. Minimal inhibition of cell proliferation (bottom graph) was observed in lowpassage cells with all three volumes of complex. In contrast, growth inhibition was observed in the high-passage cells when 6-10 µL of the complex was added. This effect on proliferation was not observed when less complex was added. (Data supplied by Roche Applied Science.)