Jul 27, 2023
Version 1
Tissue-specific in vivo transformation of plasmid DNA in Neotropical tadpoles using electroporation V.1
Peer-reviewed method
- Jesse Delia1,
- Maiah Gaines-Richardson1,
- Sarah C. Ludington1,
- Najva Akbari1,
- Cooper Vasek2,
- Daniel A. Shaykevich1,
- Lauren A O'Connell1
- 1Stanford University;
- 2De Anza College
External link: https://doi.org/10.1371/journal.pone.0289361
Protocol Citation: Jesse Delia, Maiah Gaines-Richardson, Sarah C. Ludington, Najva Akbari, Cooper Vasek, Daniel A. Shaykevich, Lauren A O'Connell 2023. Tissue-specific in vivo transformation of plasmid DNA in Neotropical tadpoles using electroporation. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5jjq4l1b/v1Version created by Lauren A O'Connell
Manuscript citation:
Delia J, Gaines-Richardson M, Ludington SC, Akbari N, Vasek C, et al. (2023) Tissue-specific in vivo transformation of plasmid DNA in Neotropical tadpoles using electroporation. PLOS ONE 18(8): e0289361. https://doi.org/10.1371/journal.pone.0289361
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 19, 2023
Last Modified: July 27, 2023
Protocol Integer ID: 82172
Keywords: tadpole, brain, muscle, electroporation, plasmid
Funders Acknowledgement:
National Science Foundation
Grant ID: IOS-1827333
New York Stem Cell Foundation
Grant ID: NYSCF-R-NI58
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.
Attachments
Delia et al electrop...
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Guidelines
These parameters are a suggested starting point. Optimizing the protocol for species-specific applications to maximize transfection efficiency is recommended.
Materials
Ethyl 3-Aminobenzoate Methanesulfonate (Millipore Sigma Catalog #10521)
Sodium Bicarbonate (Millipore Sigma Catalog #S6014)
Josh's Frogs R/O Rx
Platinum Foil (Fisher Scientific Catalog #AA11509FF)
Lead Solder Wire (Amazon Catalog #B075WB98FJ)
Loctite Fun-Tak Mounting Putty Tabs (Amazon Catalog #1865809-12)
100 mm X 15 mm Petri Dishes (Fisher Scientific Catalog #FB0875713)
Serological Pipette (Fisher Scientific Catalog #12-567-600)
Electrical Tape (Fisher Scientific Catalog #19-047-280)
Micromanipulator (Sutter Catalog #MM-33)
Grass Instruments SD9 Square Pulse Stimulator
Disposable Paired 13 mm Subdermal Needle Electrodes (MFIMedical Catalog #RHL-RLSND121-1-0)
3.5” Replacement Glass Capillaries (Drummond Scientific Catalog #3-000-203-G/X)
Sutter Instrument Co P-97 (Sutter Catalog #P-P7)
Forceps (Fisher Scientific Catalog #12-000-157)
Mineral Oil (Millipore Sigma Catalog #M8410)
28 Gauge Metal Hub Blunt Point Needle (Fisher Scientific Catalog #14815616)
1 mL BD Disposable Syringe (Fisher Scientific Catalog #14-823-30)
Nanoject II Variable Volume Automatic Injector (Drummond Scientific Catalog #3-000-204)
pCMV-GFP (Addgene Catalog #11153)
Fast Green FCF (Millipore Sigma Catalog #F7258)
Kimwipes (Fisher Scientific Catalog #06-666)
Standard Disposable Transfer Pipettes (Fisher Scientific Catalog #13-711-7M)
Micro Detail Paint Brush
Stereomicroscope with a GFP filter
Safety warnings
MS‐222 is a respiratory irritant and the following personal protective equipment should be worn: labcoat, gloves and safety glasses.
Before start
Consult with your local animal ethics board prior to experimentation.
Anesthesia Preparation
Anesthesia Preparation
5m
5m
Mix 0.02g ethyl 3-aminobenzoate methanesulfonate (MS-222) and 0.08g sodium bicarbonate with 60 mL tadpole water
5m
Store at 4C for up to one week
Electrode Set-Up for Targeting Muscle Fibers
Electrode Set-Up for Targeting Muscle Fibers
25m
25m
Remove the tips from two 5 mL serological pipettes using scissors
5m
Solder two ~ 5 mm X 8 mm pieces of platinum foil to separate electrical lead wires to make an electrode
5m
Run one electrode wire through each cut serological pipette and secure it with electrical tape
2m
Construct a platform out of clay evenly spread over the top of a Petri dish
2m
Embed the anode into the clay with the foil exposed near the center of the Petri dish
5m
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.
1m
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.
2m
Set the stimulator parameters to square wave, 1 pps, 0.1 ms delay, 5 ms duration, and 30-50 V
1m
Electrode Set-Up For Targeting Brain Cells
Electrode Set-Up For Targeting Brain Cells
25m
25m
Construct a platform out of clay molded into the shape of a hill
5m
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.
1m
Remove the tips from two 1 mL serological pipettes using scissors
5m
Mount the serological pipettes side by side on the micromanipulator and fasten them together with electrical tape
5m
Run one needle electrode wire through each cut serological pipette
2m
Position the electrode needles ~ 1 mm apart and parallel to one another
Figure 4. An assembled electrode mounted on a micromanipulator.
5m
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.
1m
Injection Set-Up
Injection Set-Up
20m
20m
Pull glass capillaries using a pipette puller
5m
Break the pipette tip at an angle using forceps to create a beveled tip
2m
Backfill the pipette with mineral oil using a 28-gauge needle and 1 mL syringe
2m
Place the pipette onto the injector plunger and tighten the collet
2m
Select an injection volume of 64.4 nl and set the injection rate to slow
2m
Empty enough mineral oil to load 2 uL of plasmid DNA solution
2m
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
2m
Fill the pipette without introducing air bubbles
1m
Connect the electrode wires to the stimulator
1m
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.
1m
Electroporation
Electroporation
25m
25m
Anesthetize the tadpole by placing it in a Petri dish of room temperature 0.03% MS-222 for 5 minutes
5m
Confirm the tadpole is fully sedated by checking for movement in response to stimuli
1m
Move the tadpole to the platform with a cut transfer pipette
2m
Adjust the position of the tadpole using a paintbrush
2m
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
For experiments targeting brain cells, place the tadpole dorsal side up with its head in the depression
Orient the platform such that the head of the tadpole is facing toward the injector
1m
Lower the injector and insert the pipette into the target tissue
2m
For experiments targeting muscle fibers, insert the pipette into a tail myomere
For experiments targeting brain cells, insert the pipette into the brain ventricle
Inject the plasmid DNA with a 5-10 s interval between each injection
2m
For experiments targeting muscle fibers, deliver two injections
For experiments targeting brain cells, deliver three injections
Remove the pipette from the tadpole
1m
Orient the platform such that the head of the tadpole is facing toward the electrode
1m
Lower the electrode until it is in full contact with the target tissue
5m
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.
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.
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.
2m
For experiments targeting muscle fibers, deliver 4-8 double pulses with a 1 s interval between each set of pulses
For experiments targeting brain cells, deliver 4-10 pulses with a 1 s interval between each pulse
Transfer the tadpole to fresh tadpole water for several hours to recover
4h
Roughly 24 to 48 hours after electroporation, screen tadpoles for plasmid uptake by imaging GFP-positive cells
In Vivo Screening
In Vivo Screening
20m
20m
Anesthetize the tadpole by placing it in a Petri dish of room temperature 0.03% MS-222 for 5 minutes
5m
Move the tadpole to a new Petri dish with tadpole water and place under a stereomicroscope with a GFP filter
5m
Center the imaging field on the target tissue and capture the fluorescent image
10m
Transfer the tadpole to fresh tadpole water for several hours to recover
4h