28 Optimization of Electroporation

Using Reporter Genes

Grant R. MacGregor

Department of Cell Biology, Howard Hughes Medical Institute, Baylor College of Medicine, Houston, Texas 77030

I. Introduction

II. Materials

III. Methods

A. Adherent Cells

B. Suspension Cells

C. Notes

References

I. Introduction

During the past 5 years, electroporation has gained wide acceptance as a powerful

method with which to introduce D N A into a variety of prokaryotic and eukaryotic

cells in culture. However, a major obstacle to the successful implementat ion of this

technique is the prerequisite determination of conditions that are op t imum for the

particular cell type being used. Voltage, capacitance, medium composition, cell

density, state of cell growth , reaction temperature, and D N A concentration are but

a few of the parameters that require consideration.

Of assistance in performing this calibration are plasmids that can express a reporter

gene product following their introduction into cells in culture. Cells may be trans-

fected under varying conditions and the relative efficiency of each transfection de-

termined by monitoring the reporter gene activity. Ideally, such a reporter gene

product should be stable, innocuous to the cell in which it is being expressed, and

readily detectable even in minute quanti t ies. For these reasons, genes that encode

enzymes that can be detected using chromogenic or radiolabeled substrates have

been widely recruited. Al though there exists a variety of reporter genes, the two

Guide to Electroporation and Electrofusion

Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved. 465

466 Grant R. MacGregor

most commonly used are the bacterial genes that encode chloramphenicol acetyl

transferase (CAT; Gorman et al., 1982) and beta-galactosidase (ß-Gal ; Hall et al.,

1983).

Arguably, of the two, Escherichia coli LacZ (ß-galactosidase; EC 3 . 2 . 1 . 2 3 ; Wal -

lenfels and Wei l , 1971) is the more versatile. Extensive characterization of both the

gene and its product has led to the development of several assays for ß-Gal activity

that are simple to perform and use relatively inexpensive, nonradioisotopic reagents.

In addition, plasmid constructs are available that can be used to generate ß-Gal

expression in a wide range of mammalian cell types (MacGregor and Caskey, 1989).

For the sake of brevity only a histochemical assay will be considered here. For a

detailed description of alternative biochemical and biological assays the reader is

referred to MacGregor et al. (1990).

Of the available assays for ß-Gal activity, the in situ histochemical assay (Bondi

et al., 1982) is the most appropriate for use in performing a determination of relative

efficiencies of cellular transfection. Briefly, cells are grown to a mid-log state of

growth, trypsinized if adherent, or pelleted if in suspension, resuspended in the

medium of choice and electroporated with plasmid that encodes E. coli ß-Gal under

the control of a suitable promoter. After a period of t ime ( 2 4 - 4 8 h) to allow recovery

and expression of the reporter gene, the cells are fixed and overlayed with a stain

containing X-Gal , a chromogenic indicator of ß-Gal activity. The X-Gal (5-chloro-

4-bromo-3-indolyl-ß-D-galactoside) is hydrolyzed by ß-galactosidase to generate

galactose and soluble indoxyl, which is subsequently converted into insoluble indigo.

The indigo is deep blue in color and facilitates determination of the relative pro-

portion of cells that have taken up and expressed the plasmid construct (Fig. 1).

The other essential components of the stain are (1) sodium phosphate, which buffers

the p H of the system to favor bacterial enzyme activity over endogenous mammalian

ß-galactosidases (which have more acidic p H optima) and provides sodium ions, an

activator of ß-Gal , (2) magnesium ions, a cofactor for the enzyme, and (3) potassium

ferro- and ferricyanide, which together act as an oxidation catalyst, increasing the

rate of conversion of the soluble indoxyl molecules to insoluble indigo, thereby

enhancing cellular localization of the enzyme activity. This assay has the advantage

that it can detect a single cell expressing ß-Gal within a population of nonexpressing

cells.

Figure 1 Evaluation of electroporation efficiency using a ß-Gal expression vector. NIH 3T3 cells were electroporated with the ß-Gal expression vector pCMVß (MacGregor and Caskey, 1989) that expresses ß-Gal under the control of a human cytomegalovirus immediate early promoter. Equal numbers of cells were electroporated with varying voltage. Cells expressing E. coli ß-Gal appear as different shades of grey. In (b), although the efficiency of transfection (number of expressing cells as a percentage of the total survivors) is greater than in (a), the number of surviving cells is con-siderably lower. This illustrates the ease with which relative frequencies of transfection and cell survival can be estimated using this technique.

Chapter 28 Optimizat ion Using Reporter Genes 467

468 Grant R. MacGregor

II. Materials

1. Stock solutions of N a 2 H P 0 4 , N a H 2 P 0 4 , and M g C l 2, each 1 M, prepared in

double distilled (dd) or Millipore (milliQ) grade water. Autoclave to sterilize

and store at room temperature. Stable indefinitely.

2. Stock solutions of potassium ferrocyanide [K 4Fe(CN) 6] and potassium ferricyanide

[K 3Fe(CN) 6] , each 50 m M . Prepare in dd or mil l iQ water, filter sterilize using

a 0 . 4 5 - p M disposable filtration uni t , and store in foil-wrapped glassware (or in

dark) at 4°C. Stable for at least 3 months .

3. Stock solution of X-Gal; 20 mg/ml . Dissolve in Ν,Ν-dimethylformamide and

store in a foil-wrapped glass container ( N O T polystyrene) at — 20°C. Stable for

at least 1 year.

4. Paraformaldehyde; 4 % (CAUTION: wear a mask and gloves when handling

paraformaldehyde and prepare in a fume cupboard). Dissolve 8 g of powder in

150 ml of 0 .1 M sodium phosphate, p H 7.3 (20 m M N a H 2 P 0 4 , 80 m M

N a 2 H P 0 4 ) by stirring and heating to around 60°C. If necessary, add 10 Ν N a O H

at the rate of 1 drop every 10 s or so unti l the solution clears. Raise the volume

to 200 ml with additional 0 .1 M sodium phosphate, p H 7 . 3 , and sterilize by

filtration. Store at 4°C for up to 1 month .

5. Gluteraldehyde (Fischer) is purchased as a 2 5 % solution.

6. Phosphate-buffered saline (PBS); composition is 15 m M sodium phosphate, p H

7 .3 , 150 m M NaCl .

7. To prepare the working fixative ( 0 . 2 % gluteraldehyde/2% paraformaldehyde),

combine 49-2 ml of 0 .1 M sodium phosphate, p H 7 . 3 , 0 .8 ml of gluteraldehyde,

and 50 ml of 4 % paraformaldehyde. Store at 4°C for up to 2 weeks.

8. To prepare the X-Gal stain, mix stocks to give final concentrations of 100 mM

sodium phosphate, p H 7.3 (80 m M N a 2 H P 0 4 , 20 m M N a H 2 P 0 4 ) , 1.3 m M

MgCl 2, 3 m M potassium ferrocyanide, 3 m M potassium ferricyanide, and 1 mg/

Table 1

Quick Calculation Table for X-Gal Stain Ingredients

Stock Final

Compound concentration concentration 5 ml 10 ml 25 ml 50 ml 100 ml

N a 2H P 0 4 1 Λ1 80 mM 400 μΐ 800 μΐ 2 ml 4 ml 8 ml

N a H , P 0 4 1 M 20 mM 100 μΐ 200 μΐ 500 μΐ 1 ml 2 ml

MgCl, 1 Λ1 1.3 mM 6.5 μΐ 13 μΐ 32.5 μΐ 65 μΐ 130 μΐ

X-Gal 20 mg/ml 1 mg/ml 250 μΐ 500 μΐ 1.25 ml 2.5 ml 5.0 ml

K,Fe(CN)6 50 mM 3 mM 300 μΐ 600 μΐ 1.5 ml 3.0 ml 6.0 ml

K4Fc(CN)6 50 mM 3 mM 300 μΐ 600 μΐ 1.5 ml 3.0 ml 6.0 ml

H>0 3.65 ml 7.3 ml 18.2 ml 36.4 ml 72.8 ml

Chapter 28 Optimizat ion Using Reporter Genes 469

C. N o t e s

1. Heterogeneity of staining within cell populations has often been observed (e .g . ,

Fig. 1). This does not appear to be due to variation of cell permeability to the

X-Gal stain, but probably reflects differences in the level of expression of ß-Gal

from cell to cell.

2. Commercially available antibodies have been shown to have greater sensitivity

ml X-Gal . Filter through a 0 . 4 5 - μ Μ disposable filtration unit prior to use. (See

Table 1 for a quick guide to preparation.) Make fresh for each occasion.

III. Methods

A. A d h e r e n t Cells

1. Aspirate media from tissue culture dishes containing cells to be assayed.

2. Rinse cell monolayers twice with PBS at room temperature.

3. Overlay cells with fixative and incubate at 4°C for 10 min .

4 . Aspirate the fixative and rinse twice with PBS.

5. Overlay cells with X-Gal stain and incubate anywhere from 15 min to overnight

at 37°C in a humidified incubator. Examine cells under microscope for ß-Gal

activity.

6. Aspirate X-Gal stain and rinse plates thoroughly, three times with 7 0 % alcohol.

This rinses out the stain and inactivates ß-Gal .

Β . Suspens ion Cells

1. Pellet the cells (from 104 to 10

7) using a centrifuge.

2. Wash once with 5 ml of PBS.

3. Pellet cells and aspirate PBS.

4. Agitate the tube to disrupt the cell pellet.

5. Add two ml of fixative and incubate at 4°C for 10 min .

6. Pellet the cells, aspirate fixative, wash cells in 5 ml PBS.

7. Pellet cells, resuspend in 2 ml X-Gal stain.

8. Transfer cells to a 24-well tissue culture plate.

9. Incubate anywhere from 15 min to overnight at 37°C in a humidified incubator.

10. Examine cells under microscope for ß-Gal activity.

11. If storage is desired, aspirate X-Gal stain and rinse with 7 0 % ethanol as for

adherent cells.

470 Grant R. MacGregor

in detecting ß-Gal (MacGregor et al., 1987). However, after staining with X-

Gal, cells cannot subsequently be stained immunocytochemically.

Acknowledgment

The author is a research associate of the Howard Hughes Medical Institute.

References

Bondi, Α., Chieregatti, G., Eusebi, V., Fulcheri, E., and Bussolati, G. (1982). The use of ß-galactosidase as a tracer in immunocytochemistry. Histochemistry 76, 153—158.

Gorman, C. M., Moffat, L. F., and Howard, Β. H. (1982). Recombinant genomes which express chloramphenicol acetyl transferase in mammalian cells. Mol. Cell Biol. 2, 1044— 1051.

Hall, C. V., Jacob, P. E., Ringold, G. M., and Lee, F. (1983). Expression and regulation of E. coli LacZ gene fusions in mammalian cells. J , ΛΙο/. Appl. Genet. 2, 101-109.

MacGregor, G. R., Mogg, A. E., Burke, J. F., and Caskey, C. T. (1987). Histochemical staining of clonal mammalian cell lines expressing E. coli ß-Galactosidase indicates het-erogeneous expression of the bacterial gene. Somat. Cell Mol. Genet. 13, 253-265.

MacGregor, G. R., and Caskey, C. T. (1989). Construction of plasmids that express E. coli ß-Galactosidase in mammalian cells. Nucleic Acid Res. 17, 2365.

MacGregor, G. R., Nolan, G. P., Fiering, S., Roederer, M., and Herzenberg, L. A. (1991). Use of E. coli LacZ (ß-galactosidase) as a reporter gene. In "Methods in Molecular Biology" (E. J. Murray, ed.), Vol 7, pp. 217-235. Humana Press, Clifton, NJ.

Wallenfels, Κ., and Weil, R. (1971). ß-Galactosidase. In "The Enzymes" (P. Boyer, ed.), 3rd ed., pp. 617-663, Academic Press, New York.