No Barriers to Entry: Transfection tools get biomolecules in the door

Date: May 24, 1999Relative Transfection Efficiency Transfection Reagents Table and Electroporation Systems Table Model of the Effectene principle. The enhancer first condenses the plasmid DNA and the effectene reagent, non- liposomal lipid, subsequently coats the condensed DNA for efficient uptake into cells. Supplied by QIAGEN. Cell transformation is an essential tool for molecular, cellular, and genetic research. Introducing specific molecules--DNA, RNA, drugs, and small molecules such as calcium--into the cytoplasm or nucleus can induce phenotypic and physiological changes that range from subtle to overwhelming. By examining the effects of the exogenous molecule on the cell, scientists can trace the pathways of a wide variety of protein synthesis, enzymatic, and immune system processes. Transformation of human cell lines in vitro has shed light on the causes and progression of autoimmune diseases, aging, cancer, and other conditions prior to in vivo testing of candidate drugs. One of the most important categories of cell transformation experiments is transfection--the introduction of DNA into a recipient eukaryotic cell, with subsequent integration into the recipient cell's chromosomal DNA. Although the considerations below apply in general to transformation experiments involving any type of molecule for uptake into any suitable target cells, this article focuses on transfection into human cell lines. The measure of a successful cell transformation is high efficiency. The challenge is to maximize the number of cells that are viable after transformation and incorporate the exogenous molecule of interest in the cytoplasm or nucleus. Important factors that affect transformation efficiency include the cell type and cell line used, tissue culture conditions, properties of the exogenous molecule to be transported, the screening assay, and finally, the tool used for transformation. Primary cells are derived from tissue and are very fragile compared to passaged cell lines. Most adherent cells derived from tissues require attachment to a petri dish in order to proliferate, with the exception of hematopoietic cells. Suspension and immortalized cells can be passaged and cultured several times using primary cells. To form the suspension cells used for transfections, adherent cells are trypsinized. Trypsinizing the cells disrupts certain aspects of the cell environment and its similarity to tissue (and therefore real organs). Tissue-derived human cell lines have been studied thoroughly since the 1950s, when the first immortalized human cell line, HeLa, was cultured from a cervical carcinoma. The HeLa cell line has been used for transfection of plasmid DNA for transient expression of a reporter gene such as luciferase or green fluorescent protein (GFP). The plasmid DNA contains various promoter genes depending on the aspect of the cells being studied. HeLa cells have also been used to study the interaction of labeled antibodies against an antigen within the cell. Tumorigenic cell lines such as the MCF-7 cell line, derived from a breast tumor, are maintained as monolayers and are used to study apoptosis in breast cancer cells. Other lines such as LNCaP, hepatoma, and hepatocytes are used to study prostate cancer and hepatitis B and C. The best source for finding a specific cell line is the American Type Culture Collection (ATCC).1 Its Web site at www.atcc.org includes pages that will answer most questions. Many cell lines are also offered commercially. Cell growth factors such as cell density and the log phase of the growth curve have a critical influence on obtaining a successful transfection or transformation of cells. Cell densities of 105-107 are ideal for transfection of cells. Other known variables such as cell line, assay type, and the transformation method should be considered when starting to optimize the cell density and growth phase to obtain the highest transformation efficiency. Tissue culture conditions are usually dictated by the type of cells and the assays used to study cell function. The right choice of medium for growth and transformation can result in viable cells with high transformation efficiency. Media such as RPMI 1640 and DMEM/F12 contain the essential nutrients for cell survival in a flask or a petri dish. The nutrients include high molecular weight carbohydrates, vitamins, proteins, and amino acids that help both monolayer and suspension cells survive and proliferate. Addition of fetal calf serum and L-glutamate to growth media such as DMEM/F12 or RPMI 1640 help in maintaining the viability of the cells. Another consideration in cell transformation is the type of assay used to screen the cell-molecule interaction. The two types of assays used for DNA transfection experiments measure transient and stable DNA expression. Transient expression occurs when DNA enters the cell nucleus but does not integrate into the cell's chromosomal DNA. Transient gene expression can be analyzed 24-96 hours after the transfection procedure by isolating protein or RNA transcripts produced by the new gene. Immunoassays can also be used to detect transient gene expression. Stable expression requires homologous integration of the DNA into the cell's chromosomal DNA. In assays for stable expression, transfected cells are isolated before proliferation. Markers such as the genes encoding thymidine kinase or aminoglycoside phosphotransferase are used to select the transfected cells before further cell proliferation. Both assays require a reporter gene driven by a promoter region. Reporter genes express proteins such as luciferase, chloramphenicol acetyltransferase (CAT), and GFP to identify viable cells containing the molecule of interest. Having the correct promoter gene ensures completion of homologous recombination and integration of the gene of interest into the cell's chromosomal DNA when using stable vector expression. Cell transformation using pharmaceutical and other compounds can be assayed using radioisotopes, fluorescent tags, and other labels. Cotransfection is also common if the molecule that needs to be transported cannot accommodate a label. In cotransfection, two molecules are added simultaneously to the cells. In theory, if the molecules have similar properties, they have a similar chance of crossing the cell membrane. Thus, one molecule is used as a marker for the transport of the second molecule. Screening methods have become more user-friendly through the introduction of robotics and automation technology. High-throughput assays are currently used to screen hundreds to thousands of cell interactions with various molecules. High-throughput assays can also be used to study gene function, cell receptor interactions with agonist and antagonist molecules, and apoptosis. Before transformation or transfection can occur, the molecules have to get across the cell membrane. Cell membranes are naturally permeable to some molecules and have cell surface receptors and pore proteins for active or passive transport of others. Four categories of transformation tools are available to transport molecules that are normally prohibited from crossing the cell membrane into the cytoplasm or nucleus. Mechanical methods such as microinjection are expensive and require a great deal of technical expertise and, therefore, are not commonly used. Chemical methods include the use of calcium phosphate or DEAE-Dextran. Lipophilic methods--"lipofection"--use liposomes and other cationic species to transport DNA, RNA, or oligonucleotides into the cell cytoplasm and, in some cases, into the cell nucleus. Electroporation can be used to transport other types of molecules as well. One example is its use in the study of tumorigenic cell interactions with fluorescence-labeled taxol. It is generally preferable to use the same method of transport in both the in vitro studies for a given experiment and the corresponding in vivo trials whenever possible to reduce the number of variables. Chemical transfection was one of the earliest methods for cell transfection and has the most published methodology references of any transformation tool. The two advantages of chemical transfection are its low cost and ease of use. However, the method is limited to the transfection of plasmid DNA, and it is not ideal for transfection of primary cells. The two chemical methods for transfection with plasmid DNA involve the use of calcium phosphate and DEAE-Dextran. Briefly, cells are trypsinized, washed, and plated the day before the transfection experiment to 30-60 percent confluency. Cells are treated with calcium phosphate/DNA or DEAE-Dextran/DNA and incubated at 37° C in a CO2 incubator for 4-16 hours, depending on the cell line. In the DEAE-Dextran method, cells are treated with the DEAE-Dextran prior to addition of DNA. Because of its toxicity to cells, DEAE-Dextran is not a preferred method for stable transfection. Glycerol or DMSO (dimethyl sulfoxide) shock can be used following transfection to improve the transfection efficiency. The duration of chemical shock should be less than 5 minutes due to its toxicity to the cells. Most commercially available kits contain all the tools necessary for a chemical transfection, including a control plasmid for optimization of the cell death curve. Determining the percentage of cell death vs. transfection efficiency enables researchers to optimize conditions such as cell exposure and DNA precipitation time variability. Specific laboratory experimental procedures can be followed using Maniatis' laboratory manuals2 or the supplier's kit procedure. Lipofection (lipid-mediated transfection) and cationic moieties have been used to transport DNA, RNA, and oligonucleotides into the cell cytoplasm or nucleus. Liposomal formulations are commonly used in the pharmaceutical industry for drug delivery of some medications with fewer side effects than in conventional formulations. Gene-based therapies also use liposomal formulations for delivery of naked DNA into cells in vivo. Today, the use of liposomes for drug delivery has proven to be efficacious for diseases such as Kaposi's sarcoma and for delivery of antibiotics to a specific target. Liposomal transport of molecules for cell transformation is one test of a proposed liposomal delivery vehicle for a given drug in humans. Lipofection requires a negatively charged molecule such as DNA to form liposome/DNA or cationic moiety/DNA complexes prior to transfection. The molecular weight of the liposome is in the range of 840-2,500 daltons. Optimization of transformation efficiency is used to determine the best liposome or cationic moiety to be used for each application. Lipofection is suitable for transfecting primary cells with either circular or linear DNA for stable or transient expression, and it can be used for in vivo applications. Disadvantages include possible toxicity and the relatively high cost of kits. Company Web sites provide a variety of protocols for each cell line. Electroporation--application of an electric field to cells--creates temporary pores in the cell membrane that allow exogenous molecules to cross into the cytoplasm or nucleus. This technology, like lipofection, also has applications in the pharmaceutical industry. It was recently awarded orphan status with the FDA as a drug delivery device and is currently in Phase IIB clinical trials in patients with squamous cell carcinoma of the head and neck. Advantages to electroporation as a cell transformation tool include millisecond transfection speeds, elimination of the need for trypsinization, the ability to transport all types of molecules, and application to in vivo delivery of molecules. The disadvantages of this method include its cost and the number of variables involved in optimization. The three important parameters in electroporation are electric field strength, pulse length, and wave shape. The electric field is the voltage that is applied to a certain area where the cells are placed in a cuvette or petri dish. Pulse length is the period of time that the cells are exposed to the electric field. Similar to the other transfection methods, a cell death curve optimization in the beginning accelerates the time necessary to determine the best field strength and pulse length for transformation. Two common wave shapes used in electroporation include exponential decay and square wave. Most commercially available electroporation instruments deliver an exponential decay wave, but a few companies offer square wave instruments with varying degrees of parameter control. Accessories are also available for electroporation of adherent cells in a 96-well plate or petri dish format. Guide to Electroporation and Electrofusion3 is a good source to learn more about the technique. Once a transformation method is chosen, optimization of transformation efficiency is the next step. Ordinary results should produce 40-80 percent of viable cells containing the molecule of interest, but in many cases, higher percentages can be obtained. The table below compares the various methods discussed above for optimal transformation efficiency. Many kit manufacturers offer samples, and the electroporation companies provide demonstration equipment that you can try in your laboratory. Relative Transfection Efficiency Transfection Reagents Table and Electroporation Systems Table The author, Mitra Ostresh, can be contacted at mostresh@home.com. American Type Culture Collection (ATCC) Reference Guide, Cell Line & Hybridomas, 8th ed., 1994 (catalog). J. Sambrook et al., eds., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, NY, CSHL Press, 1989. D.C. Change et al., eds., Guide to Electroporation and Electrofusion, San Diego, Calif., Academic Press, 1992.

No Barriers to Entry: Transfection tools get biomolecules in the door

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