S. Li (ed.), Electroporation Protocols: Preclinical and Clinical Gene Medicine. 289From Methods in Molecular Biology, Vol. 423.© Humana Press 2008

Chapter 21Electroporation of Adult Zebrafish

N. Madhusudhana Rao, K. Murali Rambabu, and S. Harinarayana Rao

Abstract We generated transient transgenic zebrafish by applying electrical pulses subsequent to injection of DNA into muscle tissue of 3–6-month old adult zebrafish. Electroporation parameters, such as number of pulses, voltage, and amount of plasmid DNA, were optimized and found that 6 pulses of 40 V/cm at 15 µg/fish increased the luciferase expression by 10-fold compared with those in controls. By measuring the expression of luciferase, in vivo by electroporation in adult zebrafish and in vitro using fish cell line (Xiphophorus xiphidium A2 cells), the strength of three promoters (CMV, human EF-1α, and Xenopus EF-1α) was compared. Subsequent to electroporation after injecting DNA in the mid region of zebrafish, expression of green fluorescent protein was found far away from the site of injection in the head and the tail sections. Thus, electroporation in adult zebrafish provides a rapid way of testing the behavior of gene sequences in the whole organism.

Keywords: zebrafish, electroporation, DNA injection, GFP, luciferase

1. Introduction

Application of electrical pulses to cells or tissue causes the formation of transient pores in the cellular membranes, leading to increased cell permeability for polar solutes and electrophoresis of the DNA into the cell (1, 2).This phenomenon was exploited to deliver nucleic acids into the cells, resulting in a tool to investigate regulatory properties of gene sequences and also to alleviate disease state by inhibiting “gain-of-function” mutations or correct “loss-of-function” mutations (3). Electrotransfer of DNA into muscle tissue has become a very popular method of gene delivery because of easy access of the muscle tissue, long life span of the muscle cell, abundant blood supply, and its suitability for the production of proteins as systemic therapeutic agents (2).

Zebrafish has proven to be a useful model organism to understand vertebrate developmental biology because of the following traits: easy husbandry, large supply

290 N.M. Rao et al.

of eggs, and transparent early stages of development (4). The small size of the zebrafish allows the use of forceps electrodes to apply pulses to bring about in vivo gene transfer. Optimization of electroporation parameters, mortality, comparison of promoter strengths, and tissue distribution of expression in zebrafish were investigated.

2. Materials

1. Cell line: A2 cell line (from late embryonic tissue of Xiphophorus xiphidium was a kind gift from Dr. N. Sivakumar of University of Hyderabad). Morphologically, the A2 cells appear cuboidal and are of epithelial origin. Cells were cultured in Dulbecco Modified Eagle’s Medium supplemented with 15% fetal bovine serum, penicillin (50 µg/mL), streptomycin (60 µg/mL), and kanamycin (100 µg/mL).

2. Molecular Biology: pCMV-Luc was a gift from Dr. Robert Debs, University of California at San Diego. Dr. Suresh Kumar of National University of Singapore provided plasmids pESG and pBOS-H2b/GFP. Restriction enzymes Hind III, Xba I, EcoR1, and Not1 were procured from New England Biolabs.

3. Culturing zebrafish: Original stock of zebrafish was obtained from a licensed sup-plier. Equipment for maintaining the tanks, aerators, thermostats, fish feed (earth worms), and reproduction tanks were either purchased or fabricated locally.

4. DNA injection and electroporation: Borosilicate capillaries with Kwik-Fil feature (OD/ID, mm – 1.0/0.75, WPI, UK), needle puller (Narasighe model PC-10), tweezer type electrodes (Tweezertrodes, 0.7 cm wide and 11.5 cm long, Model 520, BTX), and electroporator (Electrosquare porator ECM 830, BTX, San Diego, CA)

5. Histology and fluorescence imaging: Locally available high quality glass slides are coated with gelatin (0.5% w/v in double distilled water (DDW) ). Photographs are taken using Contax 16Mt camera attached to a fluorescence microscope (Zeiss Axiovision).

6. Tissue lysis buffer: 8.25 mM Tris-H3PO

4, pH 7, 2 mM DTT, 2 mM 1,2-diamino-

cyclohexane N,N,N′,N′-tetracetic acid, 10% glycerol, and 1% Triton X-100.7. Cell lysis buffer: 250 mM Tris-HCl, pH 8.0, containing 0.5% NP40.8. Culture medium: Dulbecco’s Modified Eagle’s medium containing 15% FBS,

penicillin (50 µg/mL), streptomycin (60 µg/mL), and kanamycin (100 µg/mL).

3. Methods

The involvement of a large portion of the body in electroporation for gene delivery, as described here for zebrafish, has not been attempted earlier. The application of electroporation of zebrafish eggs for transgene production has been established (5). Transgene expression in adult zebrafish has been demonstrated upon muscle injec-tion (6, 7) and by electroporating the zebrafish fins (8). A portion of the muscle,

21 Electroporation of Adult Zebrafish 291

usually the thigh muscle in mice, was used for electroporation (3). Since electropo-ration is known to cause tissue damage due to leakage of cell contents, careful optimization of the electroporation parameters is required. The objective of optimi-zation is to achieve maximum gene expression with the least toxicity/death to the zebrafish. Compared with naked DNA injection, the application of electrical pulses at the site of injection enhances transgene expression several fold. Since electrical pulses are applied across the body, involving several organs of the fish, it is impor-tant to assess the extent of expression and also sensitivity of various tissues present between the electrodes.

The efficiency of transgene expression after electroporation depends on the intensity, shape, duration, and frequency of the pulse(s). In several contexts, pulse sequences such as high voltage followed by low voltage pulses were shown to be efficient. Protocols for transient expressions of transgenes are important to understand the roles of gene sequences. In vitro data on putative roles of gene sequences often needs substantiation of in vivo situations. The several fold enhance-ment of transgene expression upon electroporation in the adult organisms allows unambiguous assessment of the behavior of gene sequences.

3.1. General Methods for Culturing Zebrafish

1. Two months old zebrafish were purchased from the local licensed fish supplier and were kept in observation tanks for one week before transferring them to experimental tanks. This period also serves for acclimatization of fish to the more controlled conditions of the laboratory.

2. The fish tanks are of 40-L capacity [(2.5 L × 1 W × 1.5 H) m3] and filled with tap water. The tanks are housed in rooms maintained at 25°C by thermostat-controlled heaters. Add commercial sea salts and minerals to the stored water (50 mg of Instant Ocean per liter of water). Allow chlorine in the water to evaporate by exposing the water to air for couple of days after the addition of dechlorination drops (see Notes 1–5). Usually 50–75 fish can be maintained in each tank. Remove two-thirds of water every alternate day. This helps the removal of debris. The storage water is maintained free from contaminants by the addition of antifungal solution (Methylene blue). Change the tanks every week and clean with potassium perman-ganate solution. Feed the fish twice a day with live earthworms. Commercial fish pellets are also suitable. Discard diseased fish, which are found occasionally because of fungal infections. Early stages of infection in fish can be treated with RIDAL, an antifungal solution, for 15 min and then transferred into main tanks.

3.2. Plasmid Preparation

1. The choice of promoters is based on the need to compare a strong promoter (CMV) with other eukaryotic promoters. Three plasmids were constructed with

292 N.M. Rao et al.

each containing either CMV (pCMV-Luc), human elongation factor (EF 1α promoter, pBOS-Luc), or Xenopus EF-1α (pESG-Luc) promoter, with luciferase as the reporter gene. The choice of promoter and plasmid backbone can be replaced depending on the experiment.

2. Plasmids pCMV/GFP, pCMV/Luc, pESG/Luc, and pBOS/Luc were isolated by CsCl density gradient method. Digest the fragment from pGL-3/Luc (Promege) with Hind III and XbaI to obtain the luciferase gene (9).

3. Double digest pESGFP plasmid with Hind III and Xba I restriction enzymes and ligate the purified backbone with the luciferase insert. We have constructed the pBOS/Luc in a similar fashion except that we have used EcoRI and NotI enzymes for taking the luciferase and inserting in the pBOS to generate pBOS/Luc. Transform the plasmids in DH5α host and purify the plasmids using Qiagen plasmid purification kits. Subsequent protocols such as gel electrophoreses, gel extraction, plasmid purifications, etc. were carried out as per standard procedures (7).

3.3. Microinjection Needle Preparation

1. Several methods of injecting the DNA solution into the fish muscle were carefully tested. Because of the small size and delicate nature of the fish, we examined metal needles (36 GA), drawn from capillaries manufactured by several compa-nies for efficiency of liquid delivery (see Note 6). The efficiency of injection can be tested by injecting a deeply colored solution (any blue dye, e.g. methylene blue and blue dextran) and monitoring the leakage (see Note 7). Metal needles were not suitable since the length of the bevel causes leakage of contents.

2. After several trials, we found glass needles drawn from borosilicate capillaries (1.0 × 90 mm2 size) having the Kwik-fil feature more reliable. Draw the needles using a needle puller (Narishige Model PC-10). Needles attached to a rubber bulb with a rubber tube can be used to draw and expel the liquid (Fig. 21.1) (see Note 8). Several needles can be pulled and calibrated by drawing a concentrated colored solution to a mark and estimating the volume by measuring the adsorp-tion of the liquid in a spectrophotometer and comparing with the standard graph prepared using commercial microsyringes.

3.4. Injection of DNA into the Fish

1. Use 3–5-month old zebrafish for electroporation.2. Hold the fish gently between the fingers or in the palm as shown in Fig. 21.1.3. Clean the injection site with 70% ethanol. Preload the needles with the DNA solu-

tion (5–20 µL of pCMV-luc) and keep them ready for injection. Once the fish is immobile, the needle can be inserted at about a 45° angle to a depth of 2–3 mm in the mid region of the fish. Carefully expel the solution from the needle by pressing

21 Electroporation of Adult Zebrafish 293

the bulb gently. Withdraw the needle at a slightly different angle after waiting for a few seconds to allow the absorption of the liquid (see Notes 9–11).

3.5. Electroporation

1. Immediately after the injection, place the fish in a narrow wooden groove padded with cotton. The groove is such that the fish would be held immobile and at the same time the sides of the fish are accessible for placing the electrodes (see Note 12).

2. Deliver the electric pulses with the set parameters to the fish (see Note 13).3. Immediately place the fish in the water containing gentamycin (50 µg/mL) to

prevent any bacterial infections.4. After electroporation, keep the fish in tanks at 28°C and maintain the treated

ones like normal fish, fed daily twice with worm pellet (see Note 14).5. Illustration of highest level of transient gene expression at 6 pulses of 40 V/cm

and 15 µg of DNA (Fig. 21.2) (see Note 15).

3.6. Estimation of Luciferase Activity

1. Monitor the fish for morbidity and death for the next 48 h. Then, dissect the fish with a scalpel to separate the head, midsection, and tail region. Freeze the tissue immediately in liquid nitrogen.

2. Add 1 mL of tissue lysis buffer to the dried tissue and homogenize using a small Teflon coated homogenizer. Remove the debris by brief centrifugation (13,000 rpm at 4°C for 5 min). Monitor luciferase activity as per the instructions provided in the kit supplied by Promega. Record the light counts using a luminometer (LUMAC Biocounter M2000). Dispense 50 µL of the Luciferase

Fig. 21.1 Picture showing the handling of zebrafish for injection (left) and electroporation

294 N.M. Rao et al.

assay reagent into luminometer tubes or microcentrifuge tubes (1.5 mL). Initiate the reading by adding 5–10 µL of the lysate. The luminometer was programmed to measure the counts after a 3-s delay followed by a 15-s measurement. Use 5 µL of the cell lysate for protein estimation by modified Lowry’s method.

3.7. Protein Assay

1. The protein content in the lysate was estimated using the Bio-Rad Protein Assay kit for normalizing the luciferase activity. Prepare the assay reagent by diluting 1 volume of the dye stock with 4 volumes of DDW. The solution should appear brown and have a pH of 1.1. It is stable for weeks in a dark bottle at 4°C. Protein standards supplied by the manufacturer were used.

2. Make up the volume of 2 µL of the lysate to 20 µL with DDW. To this solution, add 1 mL of Biorad protein assay solution. Mix and incubate at room temperature for 5 min. Absorbance of the samples is taken within an hour. Measure the absorb-ance at 595 nm and calculate the protein value from the standard curve prepared on the same day (see Note 16).

3.8. Evaluation of Promoter Strength In Vivo (Zebrafish) and In Vitro (A2 Cell Lines)

1. The utility of in vivo electroporation would be beneficial if transgene expres-sions obtained in vivo match with in vitro results. For this purpose we chose

Fig. 21.2 Influence of various parameters of electroporation on reporter gene activities. Electrical pulses were applied following intramuscular injection of plasmid DNA. A. Pulse number; B. voltage strength; C. amount of plasmid DNA present in fixed volume with (open circles) and without (closed circles) electroporation. Relative luminescence units were normalized for the amount of protein. Each data point is an average of values obtained from 6–8 fish and each experiment was repeated three times. All values of reporter gene activities on electropora-tion were significantly (p < 0.01) different from the control values (with permission from BioMedCentral)

10 15 200

400

800

1200

0 20 40 60 800

200

400

RL

U/m

g p

rote

in

Voltage (V.cm-1)

RL

U/m

g p

rote

in

DNA,mg

0 2 4 6 8 10

200

400

CA BR

LU

/mg

pro

tein

No.of Pulses

21 Electroporation of Adult Zebrafish 295

three promoters viz. CMV (pCMV-Luc), human EF-1α (pBOS-Luc), or Xenopus EF-1α (pESG-Luc) promoter. In all these constructs, luciferase was the reporter gene (see Note 17). The promoter strength was tested in A2 cells, a cell line derived from late embryonic tissue from Xiphophorus xiphidium and also in adult zebrafish by the method described earlier.

2. Maintain A2 cells at 28°C in DMEM medium in a 5% CO2 environment.

Propagate by transferring into fresh medium every 48 h. Plate the cells at a density of 16,000–20,000 cells per well in a 96-well plate one day prior to transfection. This would result in ∼70% confluency.

3. Lipofectamine, an efficient cationic liposomal preparation, was used to complex the DNA and deliver the DNA inside the cells. To form a transfection complex, incubate Lipofectamine (1–3 µL) and plasmid (0.3 µg/well) together for 30 min in serum-free DMEM. Add these complexes to cells for about 3 h. Replace the medium with DMEM medium containing 10% serum.

4. Incubate cells for 24 h before estimating the reporter gene activity. After 24 h, remove the medium, wash cells with PBS, and lyse with 50 µL lysis buffer for 10 min at room temperature. Use 5 µL of cell lysate for protein estimation by modified Lowry’s method.

5. Assay reporter gene activity with these three plasmids in A2 cell line at various charge ratios of lipid to DNA. With all three constructs, the maximum reporter gene expression was obtained at a charge ratio (+/−) of 1:1 and reporter gene activity decreased either on increase or decrease of charge ratio from 1:1.

6. Estimate luciferase activity in the tissue as described in sect. 3.6.7. Figure 21.3 shows the data obtained with the expression of three different

promoters of variable strength both in vivo (electroporation) and in vitro (by lipid-mediated transfection)

1000

100R

LU

/mg

pro

tein

pCMV pESG pBOS

Fig. 21.3 Comparison of strengths of three promoters in vivo (zebrafish, light gray) and in vitro (in A2 cell lines, dark gray). The relative luminometer counts obtained with A2 cell lines were divided by 100 for comparison with the in vivo values obtained with zebrafish (with permission from BioMedCentral)

296 N.M. Rao et al.

3.9. Expression of Green Fluorescent Protein (GFP) in Fish After Electroporation

1. Electroporate zebrafish by the protocol described earlier except that the plasmid in this study was pCMVGFP. Inject zebrafish with 10 µg of pCMVGFP and electroporate by applying 6 pulses at 40 V/cm.

2. Sacrifice the fish after 2 days. Tissues around the injection site, head, and tail regions can be taken. Inject in mid-dorsal region approximately at 60% of the total length from the head. The sections for the head region and tail region are taken at least 1 cm away on either side from the point of injection (Fig. 21.4). Snap freeze the tissues in liquid nitrogen and maintain at this temperature till cryosectioning.

3. Embed the frozen tissue in O.C.T. embedding medium (Tissue-Tek® Embedding Supplies; Sakura Finney) for cryosectioning. Carefully transfer sections of 7 µm onto gelatin (0.5%) coated slides. Store the slides at −20°C until examination. Both phase-contrast and fluorescence images (490 nm) of sections

To tube

A B C D

Fig. 21.4 Effect of electroporation on GFP expression in zebrafish. Fluorescence images from control (A) and electroporated samples (B, C, and D). A is an image taken from the tissue at the site of injection. B, C, and D represent the images obtained from head, midsection, and tail region, respectively. Fluorescence from tissue sections from head and tail of control fish is similar to A (hence not shown). At least 20 fish were electroporated in different days with pCMVGFP. We observe some variation in extent of GFP expression, but the expression was always maximum in the midregion. The presented data is from one fish (with permission from BioMedCentral)

21 Electroporation of Adult Zebrafish 297

are obtained using a Zeiss Axiovison fluorescent microscope with a camera (Contax 16MT).

4. The expression levels of GFP in head, midsection, and tail region are shown in Fig. 21.4. The sections are also stained using 4′,6-diamidino-2-phenylindole (DAPI), a simple nuclear staining fluorescent dye. Dilute DAPI (1 mg/mL) solution 1:1,000 with phosphate buffered saline and add to the section. Wash off the stain after 10 min with fresh PBS.

4. Notes

1. Maintenance of temperature at 28°C in the fish tanks is essential to achieve expected growth of zebrafish.

2. A light–dark cycle (L:D) of 12 h × 12 h was used in the room where fish tanks are maintained. 3. Dechlorination of the water is very important since the municipal water supply contains vari-

able amounts of chlorine. 4. Tanks were filled with tap water 48 h before introducing the fish. Aged tap water is known to

have reduced chlorine content. 5. Standard zebrafish husbandry practices are employed. Fish were fed twice a day. For more

specific information, the following site has many useful tips: http://zfin.org/zf_info/zfbook/zfbk.html.

6. Metal needles of any gauge were not found suitable since the extent of tissue puncture is unacceptable.

7. Careful monitoring of the leakage of solution after withdrawing the needle is very critical for complete delivery of the DNA solutions. This step, in our opinion, is the most important one for reproducible results. Needles are not reused. Needles are drawn to a very fine tapering end but, at the same time, firm enough to withstand the resistance from the tissue.

8. The design of tubing and the attachment to connect the needle can be replaced with something more convenient. We normally held the fish in our left hand and the rubber bulb in the right hand. To keep the right hand free sometimes we put the tube in the mouth and blew air to expel the liquid.

9. DNA solutions of more than 20 µL were found leaky with our needles. We often found withdrawing of the needle crucial since the DNA solution might leak back along the incision made by the needle. We have practiced the injection and withdrawal by using a colored solution.

10. The zebrafish would be under stress once taken out of water, and hence the DNA injection and application of electrical pulses needs to be completed as rapidly as possible. We com-pleted our operation in less than a minute.

11. After electroporation, the fish were found to be sluggish in their mobility for a day and then recovered their normal mobility.

12. Restraining the zebrafish at the time of applying pulses is essential. 13. Voltages more than 80 V/cm were found to increase death of the fish substantially. The mor-

tality was observed to be 6, 15, 25, and 30% at 20, 40, 60, and 80 V/cm respectively. 14. Expression of a transgene was observed after one week of electroporation whenever tested. 15. Electroporation conditions of 6 pulses of 40 V/cm were also found to be efficient in another

fish, Indian carp. 16. Performing control with the cell lysis buffers in protein estimation is essential since lysis

buffer gives significant blue color, which must be corrected. 17. For isolation of plasmids, we have used plasmid purification and gel extraction kits marketed

by Qiagen. The protocols are followed as described in the manufacturer’s brochure.

298 N.M. Rao et al.

References

1. Bigey, P., Bureau, M.F., and Scherman, D. (2002) In vivo plasmid DNA electrotransfer. Curr. Opin. Biotechnol. 13, 443–447.

2. Herweijer, H. and Wolff, J.A. (2003) Progress and prospects: naked DNA gene transfer and therapy. Gene Ther. 10, 453–458.

3. Fattori, E., La Monica, N., Ciliberto, G., and Toniatti, C. (2002) Electro-gene-transfer: a new approach for muscle gene delivery. Somat. Cell Mol. Genet. 27, 75–83.

4. Neumann, C.J. (2002) Vertebrate development: a view from the zebrafish. Semin. Cell Dev. Biol. 13, 469.

5. Powers, D.A., Hereford, L., Cole, T., et al. (1992) Electroporation: a method for transferring genes into the gametes of zebrafish (Brachydanio rerio), channel catfish (Ictalurus puncta-tus), and common carp (Cyprinus carpio). Mol. Mar. Biol. Biotechnol. 1, 301–308.

6. Sudha, P.M., Low, S., Kwang, J., and Gong, Z. (2001) Multiple tissue transformation in adult zebrafish by gene gun bombardment and muscular injection of naked DNA. Mar. Biotechnol. (NY) 3, 119–125.

7. Tan, J.H. and Chan, W.K. (1997) Efficient gene transfer into zebrafish skeletal muscle by intramuscular injection of plasmid DNA. Mol. Mar. Biol. Biotechnol. 6, 98–109.

8. Tawk, M., Tuil, D., Torrente, Y., Vriz, S., and Paulin, D. (2002) High-efficiency gene transfer into adult fish: a new tool to study fin regeneration. Genesis. 32, 27–31.

9. Sambrook, J., Fritsch, E.F., and Maniatis, T. (eds.) (1989) Molecular cloning. A laboratory manual, part II. Cold Spring Harbor Laboratory Press, New York, NY.