TITLE: Tissue-specific in vivo transformation of plasmid DNA in Neotropical tadpoles using electroporation
AUTHORS: Jesse Delia, Maiah Gaines-Richardson, Sarah C. Ludington, Najva Akbari, Cooper Vasek, Daniel Shaykevich, Lauren A O'Connell

[DESCRIPTION]
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.

[GUIDELINES]
These parameters are a suggested starting point. Optimizing the protocol for species-specific applications to maximize transfection efficiency is recommended. 

[STEPS]
SECTION: Anesthesia Preparation
1. Mix 0.02g ethyl 3-aminobenzoate methanesulfonate (MS-222) and 0.08g sodium bicarbonate with 60 mL tadpole water
SECTION: Anesthesia Preparation
2. Store at 4C for up to one week
SECTION: Electrode Set-Up for Targeting Muscle Fibers
3. Remove the tips from two 5 mL serological pipettes using scissors
SECTION: Electrode Set-Up for Targeting Muscle Fibers
4. Solder two ~ 5 mm X 8 mm pieces of platinum foil to separate electrical lead wires to make an electrode
SECTION: Electrode Set-Up for Targeting Muscle Fibers
5. Run one electrode wire through each cut serological pipette and secure it with electrical tape
SECTION: Electrode Set-Up for Targeting Muscle Fibers
7. Embed the anode into the clay with the foil exposed near the center of the Petri dish
SECTION: Electrode Set-Up for Targeting Muscle Fibers
10. Set the stimulator parameters to square wave, 1 pps, 0.1 ms delay, 5 ms duration, and 30-50 V
SECTION: Electrode Set-Up For Targeting Brain Cells
11. Construct a platform out of clay molded into the shape of a hill  

SECTION: Electrode Set-Up For Targeting Brain Cells
12. Fix the clay on the top of a Petri dish 

  
SECTION: Electrode Set-Up For Targeting Brain Cells
13. Remove the tips from two 1 mL serological pipettes using scissors
SECTION: Electrode Set-Up For Targeting Brain Cells
14. Mount the serological pipettes side by side on the micromanipulator and fasten them together with electrical tape 

SECTION: Electrode Set-Up For Targeting Brain Cells
15. Run one needle electrode wire through each cut serological pipette 

SECTION: Electrode Set-Up For Targeting Brain Cells
16. Position the electrode needles ~ 1 mm apart and parallel to one another

   
SECTION: Electrode Set-Up For Targeting Brain Cells
17. Set the stimulator parameters to 10 pps, 0.1 ms delay, 15 ms duration, and 30 V
 

SECTION: Injection Set-Up
18. Pull glass capillaries using a pipette puller
SECTION: Injection Set-Up
19. Break the pipette tip at an angle using forceps to create a beveled tip
SECTION: Injection Set-Up
20. Backfill the pipette with mineral oil using a 28-gauge needle and 1 mL syringe
SECTION: Injection Set-Up
21. Place the pipette onto the injector plunger and tighten the collet
SECTION: Injection Set-Up
22. Select an injection volume of 64.4 nl and set the injection rate to slow
SECTION: Injection Set-Up
23. Empty enough mineral oil to load 2 uL of plasmid DNA solution
SECTION: Injection Set-Up
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
SECTION: Injection Set-Up
25. Fill the pipette without introducing air bubbles
SECTION: Injection Set-Up
26. Connect the electrode wires to the stimulator
SECTION: Injection Set-Up
27. Place the platform under a dissection microscope with the electrode on one side and the injector on the other

  

SECTION: Electroporation
28. Anesthetize the tadpole by placing it in a Petri dish of room temperature 0.03% MS-222 for 5 minutes
SECTION: Electroporation
29. Confirm the tadpole is fully sedated by checking for movement in response to stimuli
SECTION: Electroporation
30. Move the tadpole to the platform with a cut transfer pipette
SECTION: Electroporation
31. Adjust the position of the tadpole using a paintbrush
SECTION: Electroporation
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
SECTION: Electroporation
31.2. For experiments targeting brain cells, place the tadpole dorsal side up with its head in the depression
SECTION: Electroporation
32. Orient the platform such that the head of the tadpole is facing toward the injector
SECTION: Electroporation
33. Lower the injector and insert the pipette into the target tissue
SECTION: Electroporation
33.1. For experiments targeting muscle fibers, insert the pipette into a tail myomere
SECTION: Electroporation
33.2. For experiments targeting brain cells, insert the pipette into the brain ventricle
SECTION: Electroporation
34. Inject the plasmid DNA with a 5-10 s interval between each injection
SECTION: Electroporation
34.1. For experiments targeting muscle fibers, deliver two injections
SECTION: Electroporation
34.2. For experiments targeting brain cells, deliver three injections
SECTION: Electroporation
35. Remove the pipette from the tadpole
SECTION: Electroporation
36. Orient the platform such that the head of the tadpole is facing toward the electrode
SECTION: Electroporation
37. Lower the electrode until it is in full contact with the target tissue
SECTION: Electroporation
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

    

SECTION: Electroporation
37.2. For experiments targeting brain cells, the electrode should be touching the head on either side of the brain

 
SECTION: Electroporation
38. Deliver the electrical pulses
 

SECTION: Electroporation
38.1. For experiments targeting muscle fibers, deliver 4-8 double pulses with a 1 s interval between each set of pulses
SECTION: Electroporation
38.2. For experiments targeting brain cells, deliver 4-10 pulses with a 1 s interval between each pulse
SECTION: In Vivo Screening
41. Anesthetize the tadpole by placing it in a Petri dish of room temperature 0.03% MS-222 for 5 minutes

SECTION: Electroporation
39. Transfer the tadpole to fresh tadpole water for several hours to recover
SECTION: In Vivo Screening
42. Move the tadpole to a new Petri dish with tadpole water and place under a stereomicroscope with a GFP filter
SECTION: In Vivo Screening
43. Center the imaging field on the target tissue and capture the fluorescent image
SECTION: In Vivo Screening
44. Transfer the tadpole to fresh tadpole water for several hours to recover
SECTION: Electroporation
40. Roughly 24 to 48 hours after electroporation, screen tadpoles for plasmid uptake by imaging GFP-positive cells
SECTION: Electrode Set-Up for Targeting Muscle Fibers
6. Construct a platform out of clay evenly spread over the top of a Petri dish
SECTION: Electrode Set-Up for Targeting Muscle Fibers
8. Create a tadpole-sized (~ 1 cm) impression adjacent the anode foil

  
SECTION: Electrode Set-Up for Targeting Muscle Fibers
9.  Mount the cathode on a micromanipulator and bend the foil parallel to the anode