Stable Transfection Of Mammalian Cells With Sense Dna Constructs

Method A. Transfection by Liposomes

Detailed protocols are described under Transient Transfection in Chapter 9. Method B. Transfection by Electroporation

The principle underlying this simple, fast and effective method is that cells and DNA constructs to be transferred are mixed and subjected to a high-voltage electrical pulse that creates pores in the plasma membranes of the cells. This allows the exogenous DNA to enter the cytoplasm of the cell and the nucleus through the nuclear pores and become integrated into the genome. This method is suitable for transient and stable transformation experiments; however, the pulse conditions should be adjusted empirically for the particular cell type. In spite of the fact that this method has a high efficiency of transfection, its drawback is that approximately 50% of the cells are killed by the high-voltage electrical pulse. These dead cells should be removed on a daily basis in order to reduce toxicity.

1. Linearize the DNA constructs to be transferred by a unique restriction enzyme. Protocols for appropriate restriction enzyme digestion are described under Subcloning of Gene and DNA Fragments in Chapter 4.

Tips: (1) The unique restriction enzyme cut should be in the plasmid backbone area such as ColE1, fi ori or ampicillin gene. It is extremely important that this cut not be in any region that will affect gene targeting by homologous recombination. The activities of Lac Z and selectable marker genes such as neo, hyg, hyrt and HSV-tk should not be affected. In the case of doubleknockout rescue DNA, ensure that the cut is not in any area in the promoter, inserted cDNA, or the splicing site. (2) In our experience, plasmid DNA constructs do not need to be linearized for homologous recombination. In fact, plasmid DNA enhances the transfection efficiency up to 30%.

2. Harvest the cells by gentle trypsinization as described in Chapter 16. Centrifuge the cells at 150 x g for 5 min and resuspend the cells well in normal culture medium at about 106 cells/ml.

Note: Make sure that the cells are gently but well suspended prior to trans-fection because aggregation of cells may cause difficulty in isolation of stably transfected cell clones.

3. In a laminar flow hood, gently mix 0.6 ml of the cell suspension with 15 to 30 ml of DNA constructs (25 to 50 mg) in a disposable electroporation cuvette (Bio-Rad, Hercules, CA).

4. According to the manufacturer's instructions, carry out electroporation using a Gene Pulser (Bio-Rad) with a pulse controller and capacitance extender at 300 V and 250 mF.

5. The electroporated cells are immediately plated in culture dishes at a density of about 3000 cells/100-mm Petri dish and cultured at 37°C with 5% CO2. Proceed to drug selection.

Method C. Transfection by Retrovirus Vectors

Detailed protocols are described in Method B under Transient Transfection.


As shown in Figure 8.1, foreign DNA enters the cells after transfection, but only a small portion of the DNA gets transferred to the nucleus in which some of the DNA might be transiently expressed for a few days. A smaller portion of the DNA introduced into the nuclei of some of the transfected cells undergoes integration into the chromosomes of the host and will be expressed in future generations of the cell population, generating stably transfected cells. The selection of the transfected cells depends on the drug-selectable marker gene fused in the recombinant plasmid, linear DNA construct or a separate plasmid that can be cotransfected into the cells. Generally, the transfected cells are cultured for 30 to 60 h in the medium lacking specific antibiotics in order to allow the cells to express the selectable-marker gene. The cells are then subcultured in a 1:10 to 100 dilution in a selective medium containing an appropriate drug. The cells are cultured in the selective medium for 10 to 14 days with changes of medium once every 2 to 7 days, depending on specific cell lines, to remove dead cells and cellular debris. In theory, only those cells that bear and express the drug-selectable marker gene can survive in the selective medium. The common marker genes that have been used for transfection and the selection can be carried out as follows.

Use of Aminoglycoside Antibiotics

If cells are transfected with recombinant gene constructs that contain a bacterial gene neomycin or Tn60 for aminoglycoside 3'-phosphotransferase (aph), the stably transfected cells can confer resistance to aminoglycoside antibiotics such as kana-mycin (kan) and geneticin (G418). For selection, a killing curve should first be established from nontransfected cells cultured in the medium containing different concentrations of an appropriate drug. Choose the concentration that kills 100% of the cells in 5 days as the concentration for selection of stably transfected cell. On the other hand, the concentration used for selection can be found from previous studies using the same cell line. For instance, we use 250 mg/ml and 1 mg/ml G418 to select mouse ES cells and fibroblast STO cells, respectively.


Stably transfected cells containing the hygromycin-P-phosphotransferase gene can be selected with hygromycin B, which is a protein synthesis inhibitor. Hygromycin B stocks are available from Sigma Chemical Co. 10 to 500 mg/ml is used in the culture medium, depending on the prekilling curve of a specific cell line.

Thymidine Kinase

If cells lacking the thymidine kinase gene (tk-) are transfected by recombinant gene constructs containing the tk gene, the stable transformants can be selected by cul-turing the transfected cells in a medium containing hypoxanthine, aminopterin, and thymidine (HAT). Nonstable transfected cells cannot metabolize this lethal analog and will be killed. The HAT medium is prepared as follows:

Dissolve 15 g hypoxanthine and 1 mg aminopterin in 8 ml of 0.1 N NaOH solution. Adjust the pH to 7.0 with 1 N HCl and then add 5 mg thymidine. Add water to 10 ml and sterile-filter it. Dilute the stock HAT to 100-fold in the culture medium used for selection. Again, the dilution or concentration of the HAT should be based on the prekilling curve. Different cell lines confer different degrees of resistance.

Tryptophan Synthetase

If cells are transfected with DNA constructs containing the tryptophan synthetase gene (the trp B), the stably transfected cells can be selected by culturing the cells in a medium lacking tryptophan, an essential amino acid necessary for the growth of cells.


Characterization of stably transfected cell lines will provide information about whether the developed clones contain and express the genes introduced into the cells. This verification step is required because drug-resistant cells may not contain or express the gene of interest. Traditionally, characterization begins with DNA analysis by Southern blotting, proceeds to the mRNA level by northern blotting, followed by protein analysis by western blotting. Nonetheless, in practice, the putative clones that express the cDNA introduced are only approximately 10% of the stably selected cell clones. Obviously, if one starts from the Southern blot analysis, there will be a tremendous amount of work, assuming that 100 clones have been obtained after drug selection and only 10 or fewer clones really express the cDNA or gene of interest. In our experience, Southern blotting as the first step of analysis may not be a good idea because it is time consuming and costly.

The same situation prevails, however, if one begins with northern blotting, which is a relatively complicated procedure. We strongly recommend starting with protein analysis by dot blotting or western blot analysis using antibodies that are specific against the protein expressed from the introduced cDNA or gene. Keep in mind that the bottom line of gene expression is the protein or the product of interest. We first choose western blot analysis12 because it is faster, less expensive, and a more reliable procedure compared with Southern and northern blot analyses. We have found that a majority of nonputative clones can be ignored after western blot analysis. Once the putative clones have been identified to express the protein of interest, one can go back to Southern or northern blot analysis. Therefore, this can be considered the reverse sequence to traditional characterization.

Analysis of Gene Overexpression at the Protein Level by Western Blotting

The detailed protocols for protein extraction and western blot hybridization are described in Chapter 11. This section focuses primarily on how to analyze western blot data. Assuming that the product or protein of the cDNA is 50 kDa and that monoclonal antibodies have been raised to be against the 50-kDa protein (Figure 8.10) in control cells (lane 1), only a weak 50-kDa band is detected. This is the endogenous level of the protein in the cells. In contrast, putative clones show overexpression of the 50-kDa protein (lanes 2 to 4) compared with the control cells.

Examination of the Expression of Sense RNA by Northern Blotting

The detailed protocols for RNA isolation and northern blot hybridization are described in Chapter 10. The major focus of this section is on how to analyze northern blot data. Assuming that the mRNA of the gene is 1 kb and that specific oligonu-cleotides or the cDNA is labeled as the probe (Figure 8.11), in control cells (lane 1), only a weak 1-kb band is detected. This is the endogenous mRNA level in the cells. In contrast, putative clones show overexpression of the mRNA (lanes 2 to 4) compared with the control cells.

Determination of Integration Copy Number by Southern Blot Analysis

Isolation of Genomic DNA from Cultured Cells

1. Aspirate the culture medium from cultured cells and rinse the cells twice in PBS.

2. Add 10 ml/60-mm culture plate (with approximately 105 cells) of DNA isolation buffer to the cells and allow the cells to be lysed for 5 min at room temperature with shaking at 60 rpm.

kDa 150 "

Protein of interest

FIGURE 8.10 Protein analysis by western blotting. Lane 1: nontransfected cells. Lane 2 to lane 4: overexpression of the 50-kDa protein in different cell clones.

Kb 10

mRNA of interest

FIGURE 8.11 mRNA analysis by northern blotting. Lane 1: nontransfected cells. Lane 2 to lane 4: overexpression of the 1-kb mRNA in different transfected cell clones.

3. Incubate the plate at 37°C for 5 h or overnight to degrade proteins and extract the genomic DNA.

4. Allow the lysate to cool to room temperature and add three volumes of 100% ethanol (precold at -20°C) to the lysate. Gently mix it and allow the DNA to be precipitated at room temperature with slow shaking at 60 rpm. Precipitated DNA, like white fibers, should be visible in 20 min.

5. "Fish" out the DNA using a sterile glass hook or spin it down. Rinse the DNA twice in 70% ethanol and then partially dry the it for 40 to 60 min at room temperature to evaporate the ethanol. The DNA can be directly subjected to restriction enzyme digestion without being dissolved in TE buffer or dd.H2O. We usually overlay the precipitated DNA with an appropriate volume of restriction enzyme digestion cocktail and incubate the mixture at the appropriate temperature for 12 to 24 h. The digested DNA is mixed with an appropriate volume of DNA loading buffer and is now ready for agarose gel electrophoresis.

DNA Isolation Buffer

75 mM Tris-HCl, pH 6.8 100 mM NaCl 1 mg/ml Protease K 0.1% (w/v) N-Lauroylsarcosine

Analysis of Southern Blot Data

Chapter 7 describes the detailed protocols for Southern blot hybridization. Assuming that genomic DNA is digested with a unique restriction enzyme that cuts outside the endogenous gene or cDNA introduced, and that the cDNA is labeled as the probe for hybridization, as shown in Figure 8.8 to Figure 8.12, lane 1 in the control cells contains one band (5 kb) of the gene. In lane 2, a putative clone carries one copy insertion of the cDNA introduced. In lane 3, a putative clone shows two copies of integration of the cDNA.

Expression Assay of a Reporter Gene

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