During electroporation, transient pores are created in a cell membrane when short electrical pulses (exceeding the membrane’s dielectric strength) are applied. While a pore is open, extracellular compounds in the vicinity of the pore can enter and transit into the cell interior. During conventional electroporation, a high concentration of cells and relevant molecules (dyes, proteins, genes, etc.) in suspension are introduced between two opposite electrodes. A high electric field, typically from 1 to 10 kV/cm, is applied to the electrodes to deliver the molecules into the cells (Huang et al. 2007; Rambabu et al. 2005; Lurquin 1997). Electroporation is a well-characterized method for molecule delivery into embryos of many species (Lurquin 1997; Swartz et al. 2001), although microinjection is more common for zebrafish embryos. As might be expected, compound trans-membrane permeability is highest for cells nearest to the electrodes (Rambabu et al. 2005). The amplitude and frequency of the applied potential, as well as its overall shape and total duration are usually empirically determined for each cell type; a range exists wherein membrane pores reseal themselves after delivery (Rambabu et al. 2005). Excessive field strength or long durations cause irreversible damage to the cells and the organism (Lurquin 1997).
All devices were fabricated on 4″ glass substrates (Supplementary Fig. 1). Wafers were cleaned in Piranha (1:1 H2SO4/H2O2) for 20 min followed by a 10-min rinse-dry cycle. Photoresist was then spun on, patterned into electrode geometries and soft-baked. A descum was performed for 3 min at 80 W to remove any residual traces of photoresist; this enhances the lift-off yield significantly. Titanium (500 Å) and platinum (1,000 Å) were deposited using evaporation and patterned using a lift-off process. Parts of the metal areas were then passivated with Polydimethylsiloxane (PDMS) acting as the silicone elastomer, leaving only the relevant metal shapes exposed to electric fields. This passivation can be performed by either (a) spinning and curing Sylgard® 184 and cutting out windows in the cured polymer using a sharp blade or (b) spinning (300 rpm), baking (130°C, 2 min), lithographically exposing and curing (150°C, 2 min) Dow Corning® WL-5150 photo-patternable silicone (according to manufacturer’s instructions) to pattern appropriate windows in the polymer. The resultant setup effectively shields electric fields and forms a chamber around the electrode to contain the solution (PDMS is hydrophobic) and the embryo.
A second set of multi-compound dosing devices were also constructed. Metal microfluidic channels were fabricated over the electrodes via a sacrificial release method and micropores were wet etched in the channel roof. These devices could deliver multiple chemicals simultaneously into the embryo. A detailed fabrication process is provided in Supplementary Fig. 2.
Zebrafish rearing and preparation
Zebrafish embryos obtained from the Duan lab were grown according to the procedure in (Westerfield 2000), maintained on a 14–10 h light dark cycle. A stock solution of E3 medium was prepared (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4, and 0.1% methylene blue) and once embryos were obtained subsequent to mating (usually daily), they were transferred in this medium and incubated at 28.5°C. Embryos were de-chorionated by mixing in 125 μg/ml of Pronase (Roche) in E3 medium and gently shaking the Petri-dish until the chorion came apart. They were rinsed thrice to remove all the pronase. Just before electroporation, embryos were anaesthetized by using MS222 (Sigma). During electroporation, embryos were held immobile within the PDMS wall chamber. They were gently pippetted into that chamber and positioned in the proper orientation over the stimulating electrodes using 5A tweezers and gentle pushing or using rolling motions. Methyl cellulose (Sigma) was also tested to hold embryos immobile, but slowed throughput and did not provide a significant advantage toward proper positioning of the embryo with respect to the electrodes.
For fluorescent imaging and dosing, solutions were prepared by mixing E3 medium with appropriate dyes. These dyes were chosen due to their low molecular weight. 30 mM solutions of Texas Red® Dextran 3,000 MW, lysine fixable (Invitrogen) and Fluorescein were prepared. A 0.4% (by volume) trypan blue (Gibco) solution was also prepared.
DNA and mRNA preparation
pCS2eGFP vectors were prepared according to the procedure outlined by Dave Turner and Ralph Rupp (Rupp et al. 1994; Turner and Weintraub 1994). Briefly, A DNA fragment containing the Kozak sequence followed by entire ORF of EGFP (Clontech, USA) was generated by PCR. This DNA fragment was subcloned into the pCS2+ vector to generate the EGFP overexpression plasmid DNA (pCS2+EGFP). The purified plasmid was dissolved in DNase free water and stored at −20°C until use. The pCS2+EGFP plasmid was linearized by restriction enzyme (NotI) digestion and was used for capped EGFP mRNA synthesis. Capped RNA was synthesized by in vitro mRNA transcription using mMassage mMachine kit (Ambion, TX). Prepared EGFP mRNA was dissolved in diethylpyrocarbonate (Sigma, USA) treated water and kept at −80°C until use.
GFP-DNA and GFP-mRNA imaging was done by Leica MZ 16F upright microscope. Adobe Photoshop and ImageJ were used to process figures. Upright and inverted microscopy was done with ZEISS Axioshop and a Nikon TE2000, respectively for dye imaging. Electroporation potential was applied with an Agilent 33220A Function/Arbitrary Waveform Generator, 20 MHz. Single square wave pulses of 10–20 V, 50–100 ms pulse width was applied between the electrodes.
Figure 1 illustrates the experimental setup. During conventional electroporation in most organisms, electrodes are placed on both sides of the embryo and transient pores are created by providing electrical pulses between them. This creates pores in the entire organism. In zebrafish embryos, however, microinjection is first used to deliver extracellular compounds locally (Fig. 1(a)) followed by electroporation of the entire embryo to enhance the microinjection results.
In our setup, lithographically-patterned metal (Fig. 1(b)) on thin glass acted as the anode and a distant metal wire was placed in solution approximately 500 μm away and acted as the cathode (these roles were switched during negatively charged pCS2eGFP DNA electroporation delivery). PDMS was used both as an insulator and as a boundary around the anode to create a chamber and contain the movement of the embryo.
During an experiment, the chamber was filled with embryo medium mixed with the appropriate delivery molecule. In the case of multi-compound dosing devices, the microfluidic channels were first filled with the appropriate dyes (and no compound was introduced into the bulk medium). Embryos were then pipetted into the chamber where they settled on top the electrode. The cathode was lowered on top of the embryo at this time. A commercial voltage source (Agilent 33220A) was used to deliver square wave pulses of 10–20 V, 50–100 ms pulse width between the electrodes. After the pulse was applied and electroporation conducted, embryos were gently removed from the device with a pipette and moved into a Petri dish containing E3 medium and incubated in 28.5°C for 24 h.