Chapter 12: Transfection

Introduction

The process of introducing nucleic acids into eukaryotic cells by nonviral methods is defined as “transfection”. Using various chemical, lipid or physical methods, this gene transfer technology is a powerful tool for studying gene function in the context of a cell. The development of reporter gene systems and selection methods for stable maintenance and expression of transferred DNA have greatly expanded the applications for transfection. Assay-based reporter technology, together with the availability of transfection reagents, provides the foundation for studying mammalian promoter and enhancer sequences, trans-acting proteins such as transcription factors, mRNA processing, protein:protein interactions, translation and recombination events (Groskreutz and Schenborn, 1997).

Essentially, transfection is a method that neutralizes or obviates the issue of introducing negatively charged molecules (e.g., phosphate backbones of DNA and RNA) into cells with a negatively charged membrane. Chemicals like calcium phosphate and DEAE-dextran or cationic lipid-based reagents coat the DNA, neutralizing or even creating an overall positive charge to the molecule (Figure 12.1). This makes it easier for the DNA:transfection reagent complex to cross the membrane, especially for lipids that have a “fusogenic” component, which enhances fusion with the lipid bilayer. Physical methods like microinjection or electroporation simply punch through the membrane and introduce the DNA directly into the cytoplasm. Each of these transfection technologies is discussed in the following sections.

This chapter covers general information on transfection techniques and considerations for transfection efficiency and optimization. In addition, we discuss the various transfection agents available from Promega as well as general protocols for transfection and specific examples using our transfection reagents. Finally, we review stable transfection and outline a protocol using drug selection.

Chemical Reagents

One of the first chemical reagents used for transfer of nucleic acids into cultured mammalian cells was DEAE-dextran (Vaheri and Pagano, 1965; McCutchan and Pagano, 1968). DEAE-dextran is a cationic polymer that tightly associates with negatively charged nucleic acids. An excess of positive charge, contributed by the polymer in the DNA:polymer complex, allows the complex to come into closer association with the negatively charged cell membrane. Uptake of the complex is presumably by endocytosis. This method is successful for delivery of nucleic acids into cells for transient expression; that is, for short-term expression studies of a few days in duration. However, this technique is not generally useful for stable or long-term transfection studies that rely upon integration of the transferred DNA into the chromosome (Gluzman, 1981). Stable transfection requires several weeks for selection, cloning and characterization of the transformed cells. Other synthetic cationic polymers have been used for the transfer of DNA into cells, including polybrene (Kawai and Nishizawa, 1984), polyethyleneimine (Boussif et al. 1995) and dendrimers (Haensler and Szoka, 1993; Kukowska-Latallo
et al. 1996).

Calcium phosphate co-precipitation became a popular transfection technique following the systematic examination of this method in the early 1970s (Graham and van der Eb, 1973). The authors examined the performance of various cations and the effect of cationic concentration, phosphate concentration and pH on transfection. Calcium phosphate co-precipitation is widely used because the components are easily available and inexpensive, the protocol is easy-to-use, and many different types of cultured cells can be transfected. The protocol involves mixing DNA with calcium chloride, adding this in a controlled manner to a buffered saline/phosphate solution and allowing the mixture to incubate at room temperature. The controlled mixing generates a precipitate that is dispersed onto the cultured cells. The precipitate is taken up by the cells via endocytosis or phagocytosis. Calcium phosphate transfection is routinely used for both transient and stable transfection of a variety of cell types. In addition, calcium phosphate also appears to provide protection against intracellular and serum nucleases (Loytner et al. 1982).

Both chemical transfer methods are relatively inexpensive and can provide high efficiency of transfer in some cell types. However, these reagents can be quite toxic (particularly DEAE-dextran), are prone to variability and are not suited for in vivo gene transfer to whole animals. In addition, small pH changes (± 0.1) can compromise the efficacy of calcium phosphate transfection (Felgner, 1990). Promega has the calcium phosphate reagent available as part of our ProFection^® Mammalian Transfection System—Calcium Phosphate (Cat.# E1200).

Cationic Lipids

By 1980, artificial liposomes were being used to deliver DNA into cells (Fraley et al. 1980). The next advance in liposomal vehicles was the development of synthetic cationic lipids by Felgner and colleagues (Felgner et al. 1987). The cationic head group of the lipid compound associates with negatively charged phosphates on the nucleic acid. Liposome-mediated delivery offers advantages such as relatively high efficiency of gene transfer, ability to transfect certain cell types that are resistant to calcium phosphate or DEAE-dextran, in vitro and in vivo applications, successful delivery of DNA of all sizes from oligonucleotides to yeast artificial chromosomes (Felgner et al. 1987; Capaccioli et al. 1993; Felgner et al. 1993; Haensler and Szoka, 1993; Lee and Jaenisch, 1996; Lamb and Gearhart, 1995), delivery of RNA (Malone et al. 1989; Wilson et al. 1979), and delivery of protein (Debs et al. 1990). Cells transfected by liposome techniques can be used for transient expression studies and long-term experiments that rely upon integration of the DNA into the chromosome or episomal maintenance. Unlike the DEAE-dextran or calcium phosphate chemical methods, liposome-mediated nucleic acid delivery can be used for in vivo transfer of DNA and RNA to animals and humans (Felgner et al. 1995).

A lipid with overall net positive charge at physiological pH is the most common synthetic lipid component of liposomes developed for gene delivery (Figure 12.2). Often the cationic lipid is mixed with a neutral lipid such as L-dioleoyl phosphatidylethanolamine (DOPE; Figure 12.3). The cationic portion of the lipid molecule associates with the negatively charged nucleic acids, resulting in compaction of the nucleic acid in a liposome/nucleic acid complex (Kabanov and Kabanov, 1995; Labat-Moleur et al. 1996), presumably from electrostatic interactions between the negatively charged nucleic acid and the positively charged head group of the synthetic lipid. For cultured cells, an overall net positive charge of the liposome/nucleic acid complex generally results in higher transfer efficiencies, presumably because this allows closer association of the complex with the negatively charged cell membrane. Entry of the liposome complex into the cell may occur by the processes of endocytosis or fusion with the plasma membrane via the lipid moieties of the liposome (Gao and Huang, 1995). Following cellular internalization, the complexes appear in the endosomes and later in the nucleus. It is unclear how the nucleic acids are released from the endosomes and lysosomes and traverse the nuclear membrane. DOPE is considered a “fusogenic” lipid (Farhood et al. 1995), and its role may be to release these complexes from the endosomes as well as to facilitate fusion of the outer cell membrane with the liposome/nucleic acid complexes. While DNA will need to enter the nucleus, the cytoplasm is the site of action for RNA, protein or antisense oligonucleotides delivered via the liposomes.

Promega provides a variety of transfection reagents that use cationic lipids for the delivery of nucleic acids to eukaryotic cells. These include the TransFast™ (Cat.# E2431), Tfx™ (Cat.# E1811, E2391) and Transfectam^® (Cat.# E1231, E1232) Reagents. All three reagents have a polycationic head group that is attached to a lipid backbone structure. The best transfection reagent and conditions for a particular cell type must be empirically and systematically determined because inherent properties of the cell influence the success of any specific transfection method.

Physical Methods

Physical methods for gene transfer were developed and used beginning in the early 1980s. Direct microinjection into cultured cells or nuclei is an effective although laborious technique to deliver nucleic acids into cells by means of a fine needle (Cappechi, 1980). This method has been used to transfer DNA into embryonic stem cells that are used to produce transgenic organisms (Bockamp et al. 2002) and for introducing antisense RNA into C. elegans (Wu et al. 1998). However, the apparatus is costly and the technique extremely labor-intensive, thus it is not an appropriate method for studies that require a large number of transfected cells.

Electroporation was first reported for gene transfer studies into mouse cells (Wong and Neumann, 1982). This technique is often used for cell types such as plant protoplasts that are difficult to transfect by other methods. The mechanism for entry into the cell is based upon perturbing the cell membrane by an electrical pulse, which forms transient pores that allow the passage of nucleic acids into the cell (Shigekawa and Dower, 1988). The technique requires fine-tuning and optimization for duration and strength of the pulse for each type of cell used. In addition, electroporation often requires more cells than chemical methods because of substantial cell death, and extensive optimization is often required to delicately balance transfection efficiency against cell viability. More modern instrumentation allows nucleic acid delivery to the nucleus and has been successful for transfer of DNA and RNA to primary and stem cells.

Another physical method of gene delivery is biolistic particle delivery, also known as particle bombardment. This method relies upon high velocity delivery of nucleic acids on microprojectiles to recipient cells by membrane penetration (Ye et al. 1990). This method has been successfully employed to deliver nucleic acid to cultured cells as well as to cells in vivo (Klein et al. 1987; Burkholder et al. 1993; Ogura et al. 2005). Biolistic particle delivery is relatively costly for many research applications, but the technology can also be used for genetic vaccination and agricultural applications.

Viral Methods

While transfection has been used successfully for gene transfer, the use of viruses as vectors has been explored as an alternative method for delivery of foreign genes into cells and as a possible in vivo option. Adenoviral vectors are useful for gene transfer due to a number of key features: 1) they rapidly infect a broad range of human cells and can achieve high levels of gene transfer compared to other available vectors; 2) adenoviral vectors can accommodate relatively large segments of DNA (up to 7.5kb) and transduce these transgenes in nonproliferating cells; and 3) adenoviral vectors are relatively easy to manipulate using recombinant DNA techniques (Vorburger and Hunt, 2002). Other vectors of interest include adeno-associated virus, herpes simplex virus, retroviruses and a subset of the retrovirus family, lentiviruses. Lentiviruses (e.g., HIV-1) are of particular interest because they have been well-studied, can infect quiescent cells, and can integrate into the host cell genome to allow stable, long-term expression of the transgene (Anson, 2004).

As with all gene transfer methods, there are drawbacks. For adenoviral vectors, packaging capacity is low, and production is labor-intensive (Vorburger and Hunt, 2002). With retroviral vectors, there is the potential for activation of latent disease and, if there are replication competent viruses present, activation of endogenous retroviruses and limited expression of the transgene (Vorburger and Hunt, 2002; Anson, 2004).

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General Considerations

Reagent Selection

With so many different methods of gene transfer, how do you choose the right transfection reagent or technique for your needs? Any time a new element, like a new cell line, is introduced, the optimal conditions for transfection will need to be determined. This may involve choosing a new transfection reagent. For example, one reagent may work well with HEK 293 cells, but a second reagent is a better choice when using HepG3 cells. One resource that might help identify a transfection reagent and protocol for your cell line would be the Transfection Assistant, a tool available online. A drop-down menu allows you to search our database by cell line, transfection type and transfection reagent. The conditions listed should be considered only guidelines since you may need to optimize the transfection conditions for your specific application. See Optimization of Transfection Efficiency and General Transfection Protocol for details.

Transient Expression versus Stable Transfection

Another parameter to consider is the time frame of the experiment you wish to conduct. Is it short- or long-term? For instance, determining which of the promoter deletion constructs still function as a promoter can be accomplished with a transient transfection experiment, while establishing stable expression of an exogeneously introduced gene construct will require a longer term experiment.

Transient Expression

Cells are typically harvested 24–72 hours post-transfection for studies designed to analyze transient expression of the transfected genes. The optimal time interval depends upon the cell type, research goals and the specific characteristics of expression for the transferred gene. Analysis of gene products may require isolation of RNA or protein for enzymatic activity assays or immunoassays. The method used for cell harvest will depend upon the end-product assayed. For example, expression of the firefly luciferase gene in the pGL4.10[luc2] Vector (Cat.# E6651) is generally assayed 24–48 hours post-transfection, whereas the pGL4.12[luc2CP] Vector (Cat.# E6671) with its protein degradation sequences can be assayed in a shorter time frame (e.g., 3–12 hours), depending on the research goals and the time it takes for the reporter gene to reach steady state. For more information on luminescent reporter genes like firefly luciferase, see the Protocols and Applications Guide chapter on Bioluminescent Reporters.

Stable Transfection

The goal of stable, long-term transfection is to isolate and propagate individual clones containing transfected DNA that has integrated into the cellular genome. Distinguishing nontransfected cells from those that have taken up the exogenous DNA involves selective screening. This screening can be accomplished by drug selection when an appropriate drug resistance marker is included in the transfected DNA. Alternatively, morphological transformation can be used as a selectable trait in certain cases. For example, bovine papilloma virus vectors produce a morphological change in transfected mouse CI127 cells (Sarver et al. 1981).

Before using a particular drug for selection purposes, you will need to determine the amount of drug necessary to kill the untransfected cells you are using. This may vary greatly among cell types. To design experiments using various concentrations of the drug to determine the amount needed for selection of resistant clones (i.e., kill curves), consult Ausubel et al. 1995 for further information.

When drug selection is used, cells are maintained in nonselective medium for 1–2 days post-transfection, then replated in selective medium containing the drug. The use of the selective medium is continued for
2–3 weeks, with frequent changes of medium to eliminate dead cells and debris, until distinct colonies can be visualized. Individual colonies can be isolated by cloning cylinders, selected and transferred to multiwell plates for further propagation in the presence of selective medium. Individual cells that survive the drug treatment expand into clonal groups that can be individually propagated and characterized. For a protocol for selecting transfected cells by antibiotics, see Stable Transfection.

Several different drug selection markers are commonly used for long-term transfection studies. For example, cells transfected with recombinant vectors containing the bacterial gene for neomycin phosphotransferase [e.g., pCI-neo Mammalian Expression Vector (Cat.# E1841)], can be selected for stable transformation in the presence of the neomycin analog G-418 (Cat.# V8091; Southern and Berg, 1982). Similarly, expression of the gene for hygromycin B phosphotransferase from the transfected vector [e.g., pGL4.14 [luc2/Hygro] Vector (Cat.# E6691)] will confer resistance to the drug hygromycin B (Blochlinger and Diggelmann, 1984).

An alternative strategy is to use a vector carrying an essential gene that is defective in a given cell line. For example, CHO cells deficient in expression of the dihydrofolate reductase (DHFR) gene do not survive without added nucleosides. However, these cells, when stably transfected with DNA expressing the DHFR gene, will synthesize the required nucleosides and survive (Stark and Wahl, 1984). An additional advantage of using DHFR as a marker is that gene amplification of DHFR and associated transfected DNA occurs when cells are exposed to increasing doses of methotrexate, resulting in multiple copies of the plasmid in the transfected cell (Schimke, 1988).

Type of Molecule Transfected

Plasmid DNA is most commonly transfected into cells, but other macromolecules can be transferred as well. For example, short interfering RNA (siRNA; Hong et al. 2004; Snyder et al. 2004; Klampfer et al. 2004), oligonucleotides (Labroille et al. 1996; Berasain et al. 2003; Lin et al. 2004), RNA (Shimoike et al. 1999; Ray and Das, 2004) and even proteins (Debs et al. 1990; Lin et al. 1993) have been successfully introduced into cells via transfection methods. However, conditions that may have worked for plasmid DNA will likely need to be optimized when using other macromolecules. In all cases, the agent transfected needs to be of high quality and relatively pure. Nucleic acids need to be free of proteins, other contaminating nucleic acids and chemicals (e.g., salts from oligo synthesis). Protein should be pure and in a solvent that is not detrimental to cell health. For additional information on plasmid DNA quality, see DNA Quality and Quantity.

Assay for Transfection

After the cells have been transfected, how will you determine success? Plasmids containing reporter genes can be used easily to monitor transfection efficiencies and expression levels in the cells. An ideal reporter gene product is one that is unique to the cell, can be expressed from plasmid DNA and can be assayed conveniently. Generally, reporter gene assays are performed 1–3 days after transfection; the optimal time should be determined empirically. For a discussion of our luminescent reporter gene options, see the Protocols and Applications Guide chapter on Bioluminescence Reporters. A direct test for the protein of interest, such as an enzymatic assay, may be another method to assess the success of transfection.

In the case of siRNA, success may be measured with a reporter gene, assaying mRNA (e.g., RT-PCR) or protein target levels (e.g., Western blotting). For additional siRNA-specific reporter options, see our Protocols and Applications Guide chapter on RNA Interference.

If multiple assays will be performed, make sure the techniques you choose are compatible with all assay chemistries. For example, if lysates are made from the transfected cells, the lysis buffer used ideally would be compatible with all subsequent assays. In addition, if cells are needed for propagation after assessment, make sure to retain some viable cells for passage after the assay.

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Factors Influencing Transfection Efficiency

With any transfection reagent or method, cell health, degree of confluency, number of passages, contamination, and DNA quality and quantity are important parameters that can greatly influence transfection efficiency. Note that with any transfection reagent or method used, some cell death will occur.

Cell Health

Cells should be grown in medium appropriate for the cell line and supplemented with serum or growth factors as needed for viability. Contaminated cells and media (e.g., yeast or mycoplasma) should never be used for transfection. If the cells have been compromised in any way, discard them and reseed from a frozen, uncontaminated stock. Make sure the medium is fresh if any components are unstable. Medium lacking necessary factors can negatively affect cell growth. Be sure the 37°C incubator is supplied with CO₂ at the correct percentage (usually
5–10%) and kept at 100% relative humidity.

If there are any concerns about what type of culture medium or CO₂ levels are needed for your cell line of interest, consult the American Type Culture Collection [ATCC] web site.

Confluency

As a general guideline, transfect cells at 40–80% confluency. Too few cells will cause the culture to grow poorly without cell-to-cell contact. Too many cells results in contact inhibition, making cells resistant to uptake of foreign DNA. Actively dividing cells take up introduced DNA better than quiescent cells.

Number of Passages

Keep the number of passages low (<50). In addition, the number of passages for cells used in a variety of experiments should be consistent. Cell characteristics can change over time with immortalized cell lines, and cells may not respond to the same transfection conditions after repeated passages, resulting in poor expression.

DNA Quality and Quantity

Plasmid DNA for transfections should be free of protein, RNA, chemical and microbial contamination. Suspend ethanol-precipitated DNA in sterile water or TE buffer to a final concentration of 0.2–1mg/ml. The optimal amount of DNA to use in the transfection will vary widely depending upon the type of DNA, transfection reagent, the target cell line and number of cells.

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Optimization of Transfection Efficiency

You will need to optimize specific transfection conditions to gain optimal transfection efficiencies. The important parameters to optimize in order to maximize transfection efficiencies are the charge ratio of cationic lipid transfection reagent to DNA, the amount of transfected nucleic acid, the length of time the cells are exposed to the transfection reagent and the presence or absence of serum. Reporter genes are useful for determining optimal conditions. The transfection efficiency achieved using any transfection reagents varies depending on the cell type being transfected and the transfection conditions used.

Charge Ratio of Cationic Transfection Reagent to DNA

The amount of positive charge contributed by the cationic lipid component of the transfection reagent used should equal or exceed the amount of negative charge contributed by the phosphates on the DNA backbone, resulting in a net neutral or positive charge on the multilamellar vesicles associating with the DNA. Charge ratios of 2:1 to 4:1 Tfx™ Reagent:DNA and 1:1 to 2:1 TransFast™ Reagent:DNA have worked well with various cultured cells (e.g., 293, HeLa, Jurkat and Sf9), but ratios outside of this range may be optimal for other cell types or applications. See Tfx™-20 and Tfx™-50 Reagents for the Transfection of Eukaryotic Cells Technical Bulletin #TB216 and TransFast™ Transfection Reagent Technical Bulletin #TB260 for more details.

DNA or RNA

The optimal amount of DNA or RNA to use in the transfection will vary depending upon the type of nucleic acid, the number of cells, the size of the culture dish and the target cell line used. For example, 293 cells are optimally transfected with 1µg of pGL3-Control DNA (Cat.# E1741) using Tfx™-50 Reagent at a 2:1 ratio in a 24-well plate. In contrast, the same cells are optimally transfected with 0.25µg of the same vector using TransFast™ Reagent at a 2:1 ratio in the same well size.

For adherent cells, we suggest optimizing DNA in the amounts recommended for the transfection reagents in Table 12.1.

It may not be necessary to increase the quantity of transfected DNA significantly to obtain optimal results. In fact, if the first transfection results are satisfactory, a reduced DNA quantity can be tested (while keeping the optimal reagent:DNA ratio constant). Often a range of DNA concentration is suitable for transfection. However, if the DNA concentration is below or above this range, transfection efficiencies will decrease. If there is too little DNA, the experimental response may not be present. If there is too much DNA, the excess can be toxic to cells. Calibrate the system using a test plasmid with reporter gene function.

The optimal CodeBreaker™ siRNA Transfection Reagent (Cat.# E5052, E5053) concentration for siRNA transfer may also require optimization in order to maximize the inhibitory effect on a given target gene expression balanced with the lowest cellular toxicity.

Time

The optimal transfection time is dependent upon the cell line, transfection reagent and nucleic acid used (Figure 12.4). For the first tests with a liposomal reagent, use a one-hour transfection interval. However, in optimization experiments, test transfection times from
30 minutes to 4 hours (or even overnight, depending on the reagent used). Monitor cell morphology during the transfection interval, particularly when the cells are maintained in serum-free medium because some cell lines lose viability under these conditions. The transfection time with the TransFast™ and Tfx™ Reagents is usually significantly shorter than that required with other cationic lipid compounds and can be decreased to as little as 30 minutes with certain cell lines. In addition to saving time, this shortened transfection time may significantly reduce the risk of cell death during the transfection procedure. Note that transfection of siRNA with CodeBreaker™ Reagent does not require either media changes or additions.

Serum

Transfection protocols often require serum-free conditions for optimal performance because serum can interfere with many commercially available transfection reagents. The TransFast™, Tfx™ and CodeBreaker™ Reagents can be used in transfection protocols in the presence of serum, allowing transfection of cell types or applications that require continuous exposure to serum (e.g., primary cells). Note that the best results are obtained when variability is minimized among lots of serum.

Co-Transfection and Dual-Reporter Assays

While many people use a single reporter gene for their experimental system, a dual-reporter system has distinct advantages. A second reporter gene allows expression to be normalized for transfection efficiency and cell number. Small perturbations in the growth conditions for the transfected cells can dramatically affect gene expression. A second reporter helps to determine if the effects are due to the treatment of the cells or a response from the experimental reporter.

Our Dual-Glo™ Luciferase Assay System (Cat.# E2920, E2940, E2980) is an efficient means of quantitating the luminescent signal from two reporter genes in the same sample. In this system, the activities of firefly (Photinus pyralis) and Renilla (Renilla reniformis) luciferases are measured sequentially from a single sample in a homogeneous format. In the Dual-Glo™ System, both reporters yield linear assay responses (with respect to the amount of enzyme) and no endogenous activity of either reporter in experimental host cells. In addition, the extended half-life of the reporter signals are ideal for use with multiwell assay formats.

Our various Renilla vectors can be used as control vectors when
co-transfected with any of our firefly luciferase vectors into which the promoter of interest has been cloned, or the firefly vector may be used as the control vector and the Renilla vector as the experimental construct. In a co-transfection experiment, it is important to realize that trans effects between promoters on co-transfected plasmids can potentially affect reporter gene expression (Farr and Roman, 1992). This is primarily of concern when either the control or experimental reporter vector or both contain very strong promoter/enhancer elements. The occurrence and magnitude of such effects will depend on several factors: 1) the combination and activities of the genetic regulatory elements present on the co-transfected vectors; 2) the amount and relative ratio of experimental vector to control vector introduced into the cells; and 3) the cell type transfected.

To help ensure independent genetic expression between experimental and control reporter genes, preliminary co-transfection experiments should be performed to optimize both the amount of vector DNA and the ratio of the co-reporter vectors added to the transfection mixture. Because our control Renilla vectors were designed for optimal expression, it is possible to use very small quantities of these vectors to provide low-level, constitutive co-expression of Renilla luciferase activity. This means that the ratio between firefly and Renilla luciferase vectors to test can range from 1:1 to 100:1 (or greater) to determine the optimal expression. The key to a dual-reporter system is to maximize the expression of the experimental reporter while minimizing that of the control reporter. However, the expression level of the control reporter should be three standard deviations above background in order to be significant.

Additionally, experimental treatments may sometimes undesirably affect control reporter expression. This compromises the accuracy of interpretation of experimental data; typically this occurs through sequences in the vector backbone, promoter or reporter gene itself. For this reason, different promoter elements along either the same vector backbone such as the pGL4.7 Vector series or a choice of vector backbones available with the synthetic Renilla vectors (phRL and phRG) are provided to select the most reliable co-reporter vector for your system. In fact, due to extremely complicated cellular experimental conditions, testing several vectors is sometimes required before finding the the best internal control for a particular experimental situation.

The strength of the promoter in your cell system is an important consideration. A more moderately expressing promoter like thymidine kinase [TK; e.g., pGL4.74[hRluc/TK] Vector (Cat.# E6921)] may be preferable to SV40 or CMV. Stronger promoters may exhibit more trans effects, cross-talk or regulatory problems. However, adjusting the ratio of the experimental vector to the control vector (e.g., using 100:1 or 200:1) may eliminate some of these issues.

For a discussion of other dual-reporter assays and vector offerings, see the Protocols and Applications Guide chapter on Bioluminescent Reporters and our complete vectors listing. Information on how to normalize dual-reporter assay data can be found in Promega Notes 72.

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Promega Transfection Products

The following sections discuss our various transfection reagents by product name. The ProFection^® Mammalian Transfection System is our only chemical reagent and uses calcium phosphate for transfection. TransFast™ Transfection Reagent, Transfectam^® and Tfx™ Reagents for the Transfection of Eukaryotic Cells discuss our four cationic lipid transfection reagents. CodeBreaker™ siRNA Transfection Reagent briefly covers our siRNA-specific transfection reagent.

ProFection^® Mammalian Transfection System

The ProFection^® Mammalian Transfection System— Calcium Phosphate (Cat.# E1200) is a simple system containing two buffers: CaCl₂ and HEPES-buffered saline. A precipitate containing calcium phosphate and DNA is formed by slowly mixing a HEPES-buffered phosphate solution with a solution containing calcium chloride and DNA. These DNA precipitates are then distributed onto eukaryotic cells and enter the cells through an endocytic-type mechanism. Calcium phosphate transfection may be used for the production of long-term stable transfectants, works well for transient expression of transfected genes and can be used with most adherent cell lines.

For a list of references using the ProFection^® Mammalian Transfection System in a variety of cell lines, see our online Transfection Assistant.

Special Usage Notes:

• Strontium chloride can be used in place of calcium chloride if the cells being transfected are sensitive to the high calcium concentration present in the calcium phosphate/DNA precipitate (Brash et al. 1987).
• To increase efficiency of transfection for some cell types, additional treatments such as glycerol (Frost and Williams, 1978; Wilson and Smith, 1997), dimethyl sulfoxide (DMSO; Lowy et al. 1978; Lewis et al. 1980), chloroquine (Luthman and Magnusson, 1983) and sodium butyrate (Gorman et al. 1983) may be added during incubation with the calcium phosphate/DNA precipitate. These treatments are thought to disrupt the phagocytic vacuole membrane, allowing the DNA to be released to the cytoplasm (Felgner, 1990). Since each of these chemicals is toxic to cells, the conditions for transfection of individual cell types must be carefully optimized for reagent concentration and exposure time.
• Alternatively, glycerol or DMSO may be added briefly (4–6 hours) after addition of the DNA precipitate (Ausubel et al. 1995).

TransFast™ Transfection Reagent

The TransFast™ Transfection Reagent (Cat.# E2431) is comprised of the synthetic cationic lipid, (+)-N,N [bis (2-hydroxyethyl)-N-methyl-N- [2,3-di(tetradecanoyloxy)propyl] ammonium iodide (Figure 12.5), and the neutral lipid, DOPE. Liposome reagents specifically designed for transfection applications like TransFast™ Reagent reflect a similar formulation by incorporating synthetic cationic lipids (Felgner et al. 1987), often together with the neutral lipid L-dioleoyl phosphatidylethanolamine (DOPE). The neutral lipid addition can enhance the gene transfer ability of certain synthetic cationic lipids (Felgner
et al. 1994; Wheeler et al. 1996). The term “liposome” refers to lipid bilayers that form colloidal particles in an aqueous medium (Sessa and Weissmann, 1968).

The TransFast™ Reagent is supplied as a dried lipid film that forms multilamellar vesicles upon hydration with water. The TransFast™ Transfection Reagent is designed for nucleic acid delivery to eukaryotic cells in vitro and in vivo (Bennett et al. 1997) and performs well with many cell lines including NIH/3T3, CHO, 293, K562, PC12, Jurkat and insect Sf9 cells. The TransFast™ Reagent combines the advantages of cationic liposome-mediated transfection with the features of speed and ease-of-use and can transfect cells for transient expression as well as stable expression. For a list of conditions that have been used successfully for the transfection of various cell types by TransFast™ Reagent, visit our Transfection Assistant.

Special Usage Notes:

• The TransFast™ Reagent can be used in the presence of serum, allowing transfection of cell types that are serum sensitive, such as primary cell cultures.
• Prepare the TransFast™ Reagent the day before transfection, because it needs to be frozen before its first use.
• There are separate protocols for transfection of adherent and suspension cells when using the TransFast™ Reagent.

Tfx™ Reagents for the Transfection of Eukaryotic Cells

The Tfx™ Reagents (Cat.# E1811, E2391, E2400) are a mixture of a synthetic, cationic lipid molecule, [N,N,N´,N´-tetramethyl-N,N´-bis(2-hydroxyethyl)- 2,3-di(oleoyloxy)-1,4-butanediammonium iodide] (Figure 12.6), and L-dioleoyl phosphatidylethanolamine (DOPE; Figure 12.3). Both Tfx™-50 and Tfx™-20 Reagents contain the same concentration of the cationic lipid component (1mM when the contents of each vial are resuspended in the recommended 400µl volume) but are formulated with different molar ratios of the neutral lipid component, DOPE.

The Tfx™ Reagents are supplied as dried lipid films. Upon hydration with water, these lipids form multilamellar vesicles that associate with nucleic acids and likely facilitate their transfer into cells by fusion of the vesicles with the cell membrane (Schenborn et al. 1995). The optimal transfection conditions for a specific cell type must be determined experimentally. In addition, this reagent can be used for stable transfection and is of low toxicity compared to standard reagents. Of note, Tfx™-50 Reagent can be used for in vivo transfection (Koegh
et al. 1997) and has been shown to be highly active in the presence of amniotic fluid (Douar et al. 1996), which has implications for its use in intra-amniotic injection and transfection.

Special Usage Notes:

• The Tfx™ Reagent Stock Solution needs to be prepared the day before transfection. After rehydration, heat to 65°C and then freeze overnight prior to use.
• There are separate transfection protocols for adherent and suspension cells.
• Transfection can occur in the presence of serum, enabling Tfx™ Reagents to be used with serum-sensitive cells like primary cells.

Transfectam^® Reagent for the Transfection of Eukaryotic Cells

Transfectam^® Reagent for the Transfection of Eukaryotic Cells (Cat.# E1231, E1232) is dioctadecylamidoglycyl spermine (DOGS), a synthetic, cationic lipopolyamine molecule. The spermine group is covalently attached through a peptide bond to the lipid moiety (Figure 12.7). The strong positive charge contributed by the spermine headgroup gives the molecule a high affinity for DNA (10⁵–10⁶M^–1), coating the DNA with a cationic lipid layer, which facilitates binding to the cell membrane. Transfectam^® Reagent allows efficient transfection of a wide range of eukaryotic cells (Behr et al. 1989; Loeffler et al. 1990; Barthel et al. 1993; Remy et al. 1994). Transfectam^® Reagent has been used for both stable and transient transfections, with both established cell lines and primary cell cultures, and for in vivo applications (Demeneix et al. 1994; Tsukamoto et al. 1995).

Studies suggest that the efficiency of transfection using DOGS is related to the structure of the lipid/DNA complex formed, and increasing both pH and ionic strength can increase formation of such complexes (Boukhnikachvili et al. 1997).

Special Usage Notes:

• Since Transfectam^® Reagent is dissolved in ethanol, it may be desirable to minimize the ethanol concentration applied to some cells. To do this, Transfectam^® Reagent may be dissolved with vortexing in as little as 1/10 volume of ethanol (dehydrated), incubated at room temperature for 5 minutes, and then further diluted to working concentration in water.
• There are two protocols available with this transfection reagent, one for medium with serum and one for medium without serum. We recommend using medium with no added serum for transfection, because some serum components may degrade the Transfectam^® Reagent.

CodeBreaker™ siRNA Transfection Reagent

The CodeBreaker™ siRNA Transfection Reagent (Cat.# E5052, E5053) is a proprietary formulation optimized for the efficient transfection of short interfering RNA (siRNA). This reagent facilitates efficient siRNA transfer into mammalian cells, allowing siRNA-mediated gene silencing with lower levels of cell death compared to other siRNA transfection reagents.

With the CodeBreaker™ siRNA Transfection Reagent, greater than 80% interference is observed in standard cell lines. CodeBreaker™ siRNA Transfection Reagent is easy to use. The reagent is mixed directly with siRNA and media, and the reagent/siRNA complex is directly added to cultured cells. Transfections can be performed in complete growth media, eliminating the requirement for a medium change. For additional information, see the Protocols and Applications Guide chapter on RNA Interference.

Special Usage Notes:

• The CodeBreaker™ siRNA Transfection Reagent should not be frozen; store at 2–8°C.
• The CodeBreaker™ Reagent:siRNA complex formation needs to occur in the absence of serum.
• For the cell lines that we have tested, the CodeBreaker™ Reagent shows improved transfection efficiencies when the cells are maintained in complete growth medium prior to transfection and no media change follows the transfection step.

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General Transfection Protocol

Preparation of Cells for Transfection

Trypsinization Procedure for Removing Adherent Cells

Trypsinizing cells for purposes of subculturing or cell counting is an important technique that is critical to successful cell culture. The following technique works consistently well when passaging cells.

Materials Required:

• 1X trypsin-EDTA solution
• 1X PBS or 1X HBSS
• adherent cells to be subcultured
• appropriate growth medium (e.g., DMEM) with serum or growth factors or both added
• culture dishes, flasks or multiwell plates, as needed
• hemocytometer

1. Prepare a sterile trypsin-EDTA solution in a calcium- and magnesium-free salt solution such as 1X PBS or 1X HBSS. The 1X solution can be frozen and thawed for future use, but the activity of the trypsin will decline with each freeze-thaw cycle. The trypsin-EDTA solution may be stored for up to 1 month at 4°C.

2. Remove the medium from the tissue culture dish. Add enough PBS or HBS solution to cover the cell monolayer: 2ml for a 150mm flask, 1ml for a 100mm plate. Rock the plates to distribute the solution evenly. Remove and repeat the wash. Remove the final wash. Add enough trypsin solution to cover the cell monolayer.

3. Place the plates in a 37°C incubator until the cells just begin to detach (usually 1–2 minutes).

4. Remove the flask from the incubator. Strike the bottom and sides of the culture vessel sharply with the palm of your hand to help dislodge the remaining adherent cells. View the cells under a microscope to check whether all cells have detached from the growth surface. If necessary, the cells may be returned to the incubator for an additional 1–2 minutes.

5. When all cells have detached, add media containing serum to the cells to inactivate the trypsin. Gently pipet the cells to break up cell clumps. The cells may then be counted using a hemacytometer and/or distributed to fresh plates for subculturing.

Typically, cells are subcultured in preparation for transfection the next day. The subculture should bring the cells of interest to the desired confluency for transfection. As a general guideline, plate 5 × 10⁴ cells per well in a 24-well plate or 5.5 × 10⁵ cells for a 60mm culture dish for ~80% confluency the day of transfection. Change cell numbers proportionally for different size plates (see Table 12.2).

^aThis information was calculated for Corning^® culture dishes.

^bRelative area is expressed as a factor of the total growth area of the 24-well plate recommended for optimization studies. To determine the proper plating density, multiply 5 × 10⁴ cells by this factor.

Preparation of DNA for Transfection

High-quality DNA free of nucleases, RNA and chemicals is as important for successful transfection as the reagent chosen. See the Protocols and Applications Guide chapter on DNA Purification for information about purifying transfection-quality DNA.

In the case of a reporter gene carried on a plasmid, a promoter appropriate to the cell line is needed for gene expression. For example, the CMV promoter works well in many mammalian cell lines but has little functionality in plants. The best reporter gene is one that is not endogenously expressed in the cells. Firefly luciferase, Renilla luciferase, click beetle luciferase, chloramphenicol aceyltransferase and
β-galactosidase fall into this category. Vectors for all five reporters are available from Promega. See our Reporter Vectors web page for more information on our wide array of reporter plasmids.

Optimization of Transfection

In previous sections, we discussed the factors that influence the success of transfection. Here we will present a methodology for optimizing transfection of a particular cell line with a single transfection reagent. If trying to choose among various reagents available to you, try the same optimization for each reagent. We recommend testing various amounts of transfected DNA (0.25, 0.5, 0.75 and 1µg per well in a
24-well plate) at two charge ratios of lipid reagent to DNA (2:1 and 4:1; see Technical Bulletin #TB216). This brief optimization can be performed using a transfection interval of one hour under serum-free conditions. One 24-well culture plate per reagent is required for the brief optimization with adherent cells (3 replicates per DNA amount).
Figure 12.8 outlines a typical optimization matrix.

A more thorough optimization can be performed to screen additional charge ratios, time points and effects of serum-containing medium at the amounts of DNA found to be optimal from the initial optimization study. One hour or two hours for the transfection interval is optimal for many cell lines. In some cases however, it may be necessary to test charge ratios and transfection intervals outside of these ranges to achieve optimal gene transfer.

Both DEAE-dextran and calcium phosphate work well with larger cell cultures (e.g., 100mm culture dish or T75 flask). General guidelines for DNA amount and time for transfection are given in Tables 12.3 and 12.4.

^aIf the cells are sensitive to the reagent, incubate for no more than 4 hours. Incubation time can be longer but will need to be optimized for the individual cell line.

^aSince DEAE-dextran is more cytotoxic than other transfection reagents, monitoring changes in cell conditions during transfection is important. The full 2.5 hours may not be necessary for efficient transfection. Once the optimal conditions are established, monitoring the cells is still important but not as critical as during optimization.

Since there is more than one transfection method for DEAE-dextran [the standard, which involves adding a DNA-DEAE-dextran mix to the cells (McCutchan and Pagano, 1968), or a modified protocol, which pretreats the cells with DEAE-dextran then adds the DNA (Al Moshlin and Dubes, 1973)], consider trying both alone and in combination with an additional treatment like glycerol, DMSO or chloroquine. These methods can increase the transfection efficiency of the chemical methods.

Some transfection methods require removal of medium with reagent after incubation; others do not. Read the technical literature accompanying the selected transfection reagent to learn which method is appropriate for your system. However, if there is excessive cell death during transfection, consider not only decreasing time of exposure to the transfection reagent, decreasing the amount of DNA and reagent added to cells or plating additional cells but also removing the reagent after the incubation period and adding complete medium.

Endpoint Assay

For assaying transient expression, many use lytic reporter assays like the Luciferase Assay System (Cat.# E1500) or the Beta-Glo^® Assay System (Cat.# E4720) 24 hours post-transfection. However, the assay time frame can range from 24–72 hours after transfection, depending on the level of protein expression. Reporter-protein assays use colorimetric, radioactive or luminescent methods to measure the enzyme activity present in a cell lysate. Some assays (e.g., Luciferase Assay System) require that the cells are lysed in a buffer after removing the medium, then mixed with a separate assay reagent to determine luciferase activity. Others are homogeneous assays (e.g., Beta-Glo^® Assay System) that have the lysis reagent and assay reagent in the same solution and can be added directly to cells in medium. Examine the readings from the reporter assay and determine where the greatest expression (highest reading) occurred. This is the point to use with your constructs of interest. See Figure 12.9 for a sample optimization using the Tfx™ Reagents.

Other assays include histochemical staining of the cells (determining the percentage of cells that were stained in the presence of the reporter gene substrate; Figure 12.10), or fluorescence microscopy (Figure 12.11) or cell sorting if using a fluorescent reporter like the Monster Green^® Fluorescent Protein phMGFP Vector (Cat.# E6421).

Assaying relative expression using the HaloTag^® Interchangeable Labeling Technology (Cat.# G8241) provides new options for rapid, site-specific labeling of proteins in living cells and in vitro. The ability to create labeled HaloTag^® fusion proteins with a wide range of optical properties and functions allows researchers to image and localize labeled HaloTag^® protein fusions in live- or fixed-cell populations and isolate and analyze HaloTag^® protein fusions and protein complexes. Several ligands are available for this system with new options being added regularly. For more information on this labeling technology, see the Protocols and Applications Guide chapter on Cell Labeling.

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Stable Transfection

Selection of Stably Transfected Cells

Optimization for stable transfection begins with successful transient transfection expression. However, cells should be transfected with a plasmid containing a gene for drug resistance, such as neomycin phosphotransferase (neo). As a negative control, transfect the cells using DNA that does not contain the drug resistance marker.

1. Prior to transfection, determine the killing concentration (kill curve) of the selective drug being used (Ausubel et al. 1995).

2. Forty-eight hours after transfection, trypsinize adherent cells and replate at several different dilutions (e.g., 1:100, 1:500) in medium containing the appropriate selection drug. For effective selection, the cells should be subconfluent since confluent, nongrowing cells are very resistant to the effects of antibiotics like G-418.

3. For the next 14 days, replace the drug-containing medium every
   3 to 4 days.

4. During the second week, monitor the cells for distinct “islands” of surviving cells. Drug-resistant clones can appear in 2–5 weeks, depending on the cell type. Cell death should occur after 3–9 days in cultures transfected with the negative control plasmid.

5. Transfer individual clones by standard techniques (e.g., using cloning cylinders) to 96-well plates and continue to maintain cultures in medium containing the appropriate drug.

In Table 12.5, an overview of commonly used antibiotics for selecting and maintaining stable transfectants is given.

Calculating Stable Transfection Efficiency

The following procedure may be used to determine the percentage of stable transfectants obtained.

Note: The stained cells will not be viable after this procedure.

Materials Required:

• methylene blue
• methanol
• cold deionized water
• light microscope

1. After approximately 14 days of selection in the appropriate drug, monitor the cultures microscopically for the presence of viable cell clones. When distinct “islands” of surviving cells are visible and nontransfected cells have died out, proceed with Step 2.

2. Prepare stain containing 2% methylene blue in 50–70% methanol.

3. Remove the growth medium from the cells by aspiration.

4. Add to the cells sufficient stain to cover the bottom of the dish.

5. Incubate for 5 minutes.

6. Remove the stain and rinse gently under deionized cold water. Shake off excess moisture.

7. Allow the plates to air-dry. The plates can be stored at room temperature.

8. Count the number of colonies and calculate the percent of transfectants based on the cell dilution and original cell number.

For further information on stable transfections, see Ausubel et al. 1995.

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Composition of Solutions

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References

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2. Anson, D.S. (2004) The use of retroviral vectors for gene therapy-what are the risks? A review of retroviral pathogenesis and its relevance to retroviral vector-mediated gene delivery. Genet. Vaccines Ther. 2, 9.
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14. Capaccioli, S. et al. (1993) Cationic lipids improve antisense oligonucleotide uptake and prevent degradation in cultured cells and in human serum. Biochem. Biophys. Res. Commun. 197, 818–25.
15. Cappechi, M.R. (1980) High efficiency transformation by direct microinjection of DNA into cultured mammalian cells. Cell 22, 479–88.
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