Tissue-specific in vivo transformation of plasmid DNA in Neotropical tadpoles using electroporation

Jesse Delia, Maiah Gaines-Richardson, Sarah C. Ludington, Najva Akbari, Cooper Vasek, Daniel A. Shaykevich, Lauren A O'Connell

Published: 2023-07-28 DOI: 10.17504/protocols.io.8epv5jjq4l1b/v1

Abstract

Electroporation is an increasingly common technique used for exogenous gene expression in live animals, but protocols are largely limited to traditional laboratory organisms. The goal of this protocol is to enable in vivo electroporation techniques in a diverse array of tadpole species. We explore electroporation efficiency in tissue-specific cells of five species from across three families of tropical frogs—poison frogs (Dendrobatidae), forest frogs (Aromobatidae), and glassfrogs (Centrolenidae). These species are well-known for their diverse social behaviors and intriguing physiologies that coordinate chemical defenses, aposematism, and/or transparency. Specifically, we examine the effects of electrical pulse and injection parameters on species- and tissue-specific transfection of plasmid DNA in tadpoles. After electroporation of a plasmid encoding green fluorescent protein (GFP), we found strong GFP fluorescence within brain and muscle cells that increases with the amount of DNA injected and electrical pulse number. We discuss species-related challenges, troubleshooting, and outline ideas for improvement. Extending in vivo electroporation to diverse amphibian species will offer a powerful approach to explore topics in genetics, behavior, and organismal biology.

Before start

Consult with your local animal ethics board prior to experimentation.

Attachments

Delia et al electroporation how to

Steps

Anesthesia Preparation

Mix 0.02g ethyl 3-aminobenzoate methanesulfonate (MS-222) and 0.08g sodium bicarbonate with 60 mL tadpole water

Store at 4C for up to one week

Electrode Set-Up for Targeting Muscle Fibers

Remove the tips from two 5 mL serological pipettes using scissors

Solder two ~ 5 mm X 8 mm pieces of platinum foil to separate electrical lead wires to make an electrode

Run one electrode wire through each cut serological pipette and secure it with electrical tape

Construct a platform out of clay evenly spread over the top of a Petri dish

Embed the anode into the clay with the foil exposed near the center of the Petri dish

Create a tadpole-sized (~ 1 cm) impression adjacent the anode foil

Figure 1. An anode embedded in a flat clay platform to hold tadpoles during the electroporation procedure.

Mount the cathode on a micromanipulator and bend the foil parallel to the anode

Figure 2. A cathode mounted on a micromanipulator in position to make contact directly above the anode during the electroporation procedure.

10.

Set the stimulator parameters to square wave, 1 pps, 0.1 ms delay, 5 ms duration, and 30-50 V

Electrode Set-Up For Targeting Brain Cells

11.

Construct a platform out of clay molded into the shape of a hill

12.

Fix the clay on the top of a Petri dish

Figure 3. A clay platform molded into the shape of a hill to hold tadpoles during the electroporation procedure.

13.

Remove the tips from two 1 mL serological pipettes using scissors

14.

Mount the serological pipettes side by side on the micromanipulator and fasten them together with electrical tape

15.

Run one needle electrode wire through each cut serological pipette

16.

Position the electrode needles ~ 1 mm apart and parallel to one another

Figure 4. An assembled electrode mounted on a micromanipulator.

17.

Set the stimulator parameters to 10 pps, 0.1 ms delay, 15 ms duration, and 30 V

Note

These settings are a suggested starting point. Optimizing these parameters for each species, as the best pulse shape and settings may vary, is recommended.

Injection Set-Up

18.

Pull glass capillaries using a pipette puller

19.

Break the pipette tip at an angle using forceps to create a beveled tip

20.

Backfill the pipette with mineral oil using a 28-gauge needle and 1 mL syringe

21.

Place the pipette onto the injector plunger and tighten the collet

22.

Select an injection volume of 64.4 nl and set the injection rate to slow

23.

Empty enough mineral oil to load 2 uL of plasmid DNA solution

24.

Pipette 2 uL of plasmid DNA solution (0.25–0.27μg/μl) onto a piece of parafilm and mix with 0.2 uL 0.01% Fast Green

25.

Fill the pipette without introducing air bubbles

26.

Connect the electrode wires to the stimulator

27.

Place the platform under a dissection microscope with the electrode on one side and the injector on the other

Figure 5. A dissection microscope focused on a tadpole injection platform that is placed between an electrode mounted on a micromanipulator and a microinjector mounted on a micromanipulator.

Electroporation

28.

Anesthetize the tadpole by placing it in a Petri dish of room temperature 0.03% MS-222 for 5 minutes

29.

Confirm the tadpole is fully sedated by checking for movement in response to stimuli

30.

Move the tadpole to the platform with a cut transfer pipette

31.

Adjust the position of the tadpole using a paintbrush

31.1.

For experiments targeting muscle fibers, place the tadpole flat on its side with its head in the depression and its tail on top of the anode

31.2.

For experiments targeting brain cells, place the tadpole dorsal side up with its head in the depression

32.

Orient the platform such that the head of the tadpole is facing toward the injector

33.

Lower the injector and insert the pipette into the target tissue

33.1.

For experiments targeting muscle fibers, insert the pipette into a tail myomere

33.2.

For experiments targeting brain cells, insert the pipette into the brain ventricle

34.

Inject the plasmid DNA with a 5-10 s interval between each injection

34.1.

For experiments targeting muscle fibers, deliver two injections

34.2.

For experiments targeting brain cells, deliver three injections

35.

Remove the pipette from the tadpole

36.

Orient the platform such that the head of the tadpole is facing toward the electrode

37.

Lower the electrode until it is in full contact with the target tissue

37.1.

For experiments targeting muscle fibers, the tail should lay on top of the anode and the cathode should press on the tail directly above the anode

Figure 6. Muscle electroporation in the tail of a Ranitomeya imitator tadpole.

37.2.

For experiments targeting brain cells, the electrode should be touching the head on either side of the brain

Figure 7. Brain electroporation in a Ranitomeya imitator tadpole with the electrode making contact with either side of the brain.

38.

Deliver the electrical pulses

Note

The pulse range for targeting brain cells is a suggested starting point. Optimizing the protocol for species-specific applications to maximize transfection efficiency is recommended.

38.1.

For experiments targeting muscle fibers, deliver 4-8 double pulses with a 1 s interval between each set of pulses

38.2.

For experiments targeting brain cells, deliver 4-10 pulses with a 1 s interval between each pulse

39.

Transfer the tadpole to fresh tadpole water for several hours to recover

40.

Roughly 24 to 48 hours after electroporation, screen tadpoles for plasmid uptake by imaging GFP-positive cells

In Vivo Screening

41.