Ex Ovo Electroporation of DNA Vectors into Pre-gastrulation Avian Embryos

Authors: Cheng Cui, Brenda Rongish, Charles Little and Rusty Lansford Corresponding author ([rusty@caltech.edu](rusty@caltech.edu)) ### INTRODUCTION The transfection of GFP-expressing constructs into early embryos permits key developmental events such as gastrulation to be dynamically imaged using time-lapse video-microscopy. This protocol describes the ex ovo electroporation of a DNA plasmid into avian embryos as young as stage X, nearly 24 h earlier in development than most electroporation protocols. Compared to in ovo electroporation, the ex ovo method allows easier embryo orientation (the posterior half of the embryo is darker than the anterior half). Thus, positioning of the specimen and consistency of the electroporation site between embryos is improved. Furthermore, nearly all embryos can be electroporated at the same stage using the ex ovo method: If some embryos have not reached a desired stage, it is possible to temporally stop development of those embryos already at the desired stage by keeping them at room temperature while incubating the rest at 37°C until they develop. The method described here uses relatively low voltage, and the electroporation chamber can be made easily, with no specialized equipment required. ### RELATED INFORMATION This protocol was adapted from Cui et al. (2006) and incorporates the ex ovo culture method described by New (1955). ### MATERIALS 1. Reagents - Buffered phenol red solution - DNA plasmid of interest (e.g., GFP expression plasmid) - *Store DNA plasmid stocks in endotoxin-free H2O.* - DNA purification kit (endotoxin-free) (e.g., Qiagen EndoFree Plasmid Maxi Kit, 12362) - Embryonic culture dish - *Prepare by pouring 3 mL of agar substrate into a 35-mm Petri dish (Fisher Scientific, 0875711YZ) (Chapman et al. 2001)*. - Embryonic culture insert - *Prepare by pouring 1 mL of agar substrate into a *Millicell insert (Millipore Organotypic culture inserts*, PICMORG-50)*. - Embryonic phosphate-buffered saline (ePBS) (1X) - Embryos from Japanese quail eggs (*Coturnix coturnix japonica*) or chicken eggs (unincubated or incubated to desired stage) - *Early embryos in this protocol are staged according to Eyal-Giladi and Kochav (1976)*. - H2O, endotoxin-free - Hanks balanced salt solution (HBSS) (Mediatech/cellgro, 21-020-CV) - Nail polish (clear) - Clear nail polish acts as an electrical insulator. ### Equipment 1. Coverslips (35 mm, No. 1), glass - Filter paper rings for embryo removal (see Step 8) - Forceps - Incubator at 37°C - Microcapillaries (1.0-mm O.D. with glass filament) (Narishige, GD-1) - Micromanipulators (2) (Narishige) - Petri dishes (60 mm) (BD Falcon, 353002) - Petri dishes (100 mm) (BD Falcon, 351029) - Picoliter injector (Harvard Instruments, PLI-100) - Platinum wires (3 × 0.35 mm and 80 × 0.3 mm) - Scissors, angled iris (Fine Science Tools, 14063-09) - Single-stage glass microelectrode puller (Narishige, PC10) - Square-pulse electroporator (BEX Co., Ltd., CUY-21TM) - Tissues - Transfer pipettes ### METHOD **Construction of Electroporation Chamber** *The use of a glass-bottomed chamber allows the precise placement of the specimen directly over the anode. The chamber is constructed such that the raised segment of the anode is bathed in electrolyte solution (HBSS) during electroporation. When the Millicell insert is centered over the glass window, the insert membrane touches the anode*. 1. Use a 60-mm polystyrene Petri dish to mark the base of the chamber. - Prepare a cathode (−) consisting of a 3 × 0.35-mm platinum wire. Approximately 2 mm from the end, bend the wire at a 45° angle (see Fig. 1, top electrode). Using clear nail polish, insulate the wire near the junction where the cathode will be mounted on the micromanipulator. Couple the cathode to a stainless steel rod and mount on a coarse-grade micromanipulator.  **Figure 1**. A cross-section of the glass-bottomed electroporation chamber used to electroporate pre-gastrulation stage avian embryos. *The cathode is positioned parallel to the anode during electroporation*. 3.Design an anode (+) that runs along the bottom of the dish using a platinum wire (80 × 0.3 mm). Bend the wire at the midsection to create a 3-mm segment that is raised 1 mm above the center of the dish and runs parallel to the dish floor (Fig. 1, bottom electrode). Attach the lengths of wire on either side of the raised loop to the floor of the dish using clear nail polish. 4.Create a glass-bottomed chamber by boring a 20-mm hole in the bottoms of both the 60-mm dish and a 100-mm Petri dish. Align the holes, and glue the 60-mm dish inside the 100-mm dish. Center a 35-mm No. 1 glass coverslip over the hole, and glue the glass to the underside of the 100-mm dish. **DNA Plasmid Preparation** 5.Isolate and purify a GFP-expression plasmid (or other DNA plasmid of interest) using an endotoxin-free DNA purification kit, e.g., the EndoFree Plasmid Maxi Kit (Qiagen). Store the purified stock in endotoxin-free H2O. 6.Prepare the plasmid DNA for electroporation by mixing a 5 μg/μL concentrated stock of endotoxin-free plasmid with buffered phenol red solution in a 1:1 ratio, such that the final DNA concentration is ~2.5 μg/μL. - *The phenol red allows visualization of the DNA bolus during injection, and serves as an indicator dye during electroporation (see Step 12.vi)*. **Embryo Removal Using Paper Rings** *This technique for isolating and securing chicken embryos on a paper ring is described by Chapman et al. (2001)*. 7.Gently pour the albumen and yolk (with embryo) of a fertile quail or chicken egg into a 100-mm Petri dish. Use a transfer pipette to remove the thick albumen from the poured contents. Use tissues to remove any albumen remaining on the embryo. 8.Remove the embryo from the yolk: - i. Place a paper ring around the embryo, on the vitelline membrane. - ii. Crimp the paper to the membrane by trimming the ring just inside its outer circumference using angled iris scissors. - iii. Gently grasp the ring at the posterior end of the embryo and remove the specimen. 9.With the ventral side up, gently submerge the specimen in embryo phosphate-buffered saline (ePBS) to remove any adherent yolk. - *Specimens can be stored in embryonic culture dishes at room temperature for several hours if necessary*. **Electroporation of Embryos** *With the use of Millicell inserts (in which the volume of agar substrate and thus the distance between the embryo and anode remain constant) as well as a glass-bottomed chamber (which allows the specimen to be placed directly over the anode), a specimen can be electroporated approximately every 3 min*. 10.Fill the electroporation chamber with HBSS, so that the fluid covers the anode. - *The fluid level must be high enough to be in contact with the underside of the Millicell insert membrane*. 11.Test the electrical continuity of the chamber: - i. Attach the leads to the chamber. Position the cathode parallel to the anode, at a distance of ~3 mm. - ii. Program the CUY-21TM power supply with the following pulse sequence: 5 V, 40 msec power-on, and 900 msec power-off, for a series of four square wave pulses. - *Bubbles should be visible on the cathode. An amperage value of at least 0.01 A should be displayed after the completion of the pulse series*. - iii. Decrease the voltage setting to 4 V after conductance has been established. 12.Electroporate the embryo with the DNA plasmid: - i. Place the embryo ventral side down on the center of an embryonic culture (Millicell) insert. Transfer the embryo/insert assembly into the electroporation chamber. Adjust the position of the insert so that the targeted region is centered on the anode. - ii. Break the tip of a previously pulled fine-diameter glass micro-needle with forceps, to obtain a point sharp enough to penetrate the vitelline membrane. Use the technique shown in Figure 2 to push the needle tip through the vitelline membrane at an acute angle (<45°), so that the membrane is penetrated without damage to the epiblast. Position the needle tip above the desired region of the epiblast.  **Figure 2**. A schematic showing the movement of the needle tip used to penetrate the vitelline membrane (V.M.) without damaging the epiblast. The top layer is the vitelline membrane and the lower layer is the epiblast. (A) The needle is lowered to press on the surface of the vitelline membrane. (B) The needle tip is moved to the left until a fold of vitelline membrane is formed over the needle tip. (C) The needle tip is lifted slightly to release the pressure on the epiblast, without losing the vitelline-membrane fold. (D) The needle tip is moved to the left to penetrate through the vitelline membrane. Steps B through D may be repeated until the needle tip penetrates the vitelline membrane. - iii. Set the ballast pressure of the injection apparatus to the P-balance mode. Pump 20 to 100 nL of the DNA/phenol red solution (2.5 μg/μL plasmid) into the space between the epiblast and the vitelline membrane. - *The P-balance setting allows slow (~5 sec), low-pressure delivery (<1.0 psi). Injection pressures >1.0 psi tend to displace cells and damage the embryo in early stages. Confine the plasmid bolus to a small area in the target region*. - iv. After delivery of the DNA plasmid, remove the glass needle. If necessary, adjust the position of the embryo/insert relative to the anode to correct any displacement caused by the injection. Adjust the cathode to a position parallel to the anode, as close to the specimen as possible without making contact. - v. Apply approximately one to two drops of HBSS to fill the gap between the cathode and the vitelline membrane (Fig. 3A). Lower the cathode further, until it touches the membrane and a deformation of tissue can be seen (Fig.3B). Raise the cathode only until tissue deformation is no longer visible (Fig. 3C).  **Figure 3**. A schematic showing movement of the cathode for electroporation. Red indicates the injected DNA plasmid solution (with phenol red). Pink indicates the presence of electrolyte solution (HBSS). (A) After the application of one to two drops of HBSS, the cathode is lowered. (B) The cathode is lifted when tissue deformation is seen. (C) The cathode is held in position as soon as tissue deformation is no longer visible. V.M. is vitelline membrane. - vi. Activate the power supply at 4 V. Look for the formation of bubbles at the cathode, as well as a change in the color of the indicator dye from pink to red, to confirm electrical continuity during the pulses. - *To transfect fewer cells within a target area (desired in some cases, such as cell tracking), 3 V may be used for electroporation (see Fig. 4A,B). If only a small amount of DNA plasmid is pumped to the target region, a small group of cells (~100) can be fluorescently labeled (Fig. 4C)*.  **Figure 4**. Images of GFP-expressing embryos 3.5 h post-electroporation. (A) Bright-field image of an avian embryo. (B) Enlarged fluorescent image (GFP) of the rectangle box in A, showing that fluorescently labeled cells on the right side of the embryo are sparser than those on the left side when different voltages were used (3 V on right side and 4 V on left side). (C) A small group of cells (~100) were fluorescently labeled when a small amount of plasmid DNA was pumped into the region. Scale bars: 250 μm in A, B, and C, and 50 μm in inset picture of C. - vii. Remove the cathode and the embryo/insert assembly from the electroporation chamber. 13.Gently submerge the embryo/insert in an ePBS-filled dish. With the assembly submerged, float the embryo with attached ring away from the agar bed/insert. Transfer to an embryonic culture dish with the ventral side up. - *When it is kept in a 37°C incubator, the cultured embryo normally can grow up to 40 h after electroporation, or until HH stage 12*. 14.Culture the specimen for a time sufficient to obtain detectable nuclear fluorescence, according to the vector or fluorescent protein used. - *Using the pH2B-cherry expression vector, nuclear fluorescence is detectable ~3 h after electroporation. Vectors with less active promoters or fluorescent proteins directed to a large volume will likely require more time to produce an observable fluorescent signal. Using our H2B-GFP plasmid the signal can be detected <2 h after electroporation*. ### DISCUSSION This technique, together with time-lapse imaging methods, allows researchers to study how these fluorescently labeled cells move or change their shapes during gastrulation. The technique can be extended when other reagents are used to study functionality of certain genes. For example, a specific gene can be over-expressed, and/or ectopically expressed, by electroporating a plasmid encoding the gene of interest. Alternatively, the functionality of a specific ligand can be down-regulated with the electroporation of a plasmid encoding the dominant-negative receptor for the ligand. A morpholino can also be used to interfere with a targeted gene pathway. ### ACKNOWLEDGMENTS This work was funded in part by the American Heart Association Postdoctoral Fellowship (0620105Z) to C.C. and the NIH NCRR (R21HD047347-01A2) to R.L. Previous Section ### REFERENCES 1. Chapman S.C., Collignon J., Schoenwolf G.C., Lumsden A. (2001) [Improved method for chick whole-embryo culture using a filter paper carrier](http://cshprotocols.cshlp.org/external-ref?access_num=10.1002/1097-0177(20010301)220:3%3C284::AID-DVDY1102%3E3.0.CO;2-5&link;_type=DOI). *Dev. Dyn*. 220:284–289. - Cui C., Lansford R., Filla M.B., Little C.D., Cheuvront T.J., Rongish B.J. 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