Aug 20, 2023
Version 1
Use and efficiency of morpholinos in Neotropical tadpole brains V.1
- 1Stanford University
Protocol Citation: Sarah C. Ludington, Julie M Butler, Chloe Golde, Lauren A O'Connell 2023. Use and efficiency of morpholinos in Neotropical tadpole brains. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm23y6g3p/v1
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: November 16, 2022
Last Modified: August 20, 2023
Protocol Integer ID: 72812
Keywords: amphibian, poison frog, vivo morpholinos, microinjection, electroporation, tyrosine hydroxylase
Funders Acknowledgement:
NSF EDGE
Grant ID: NSF IOS 1827333
Rita Allen
Grant ID: Scholar's Award
Abstract
Antisense morpholinos are a common tool used to knockdown protein abundance in target tissues in fish and amphibians. However, protocols are largely limited to common aquatic research organisms, such as zebrafish or Xenopus frogs. The goal of this protocol is to enable in vivo morpholino delivery, visualization, and knockdown evaluation in the Neotropical tadpoles. We tested the efficiency of standard and Vivo-morpholinos in the knockdown of tyrosine hydroxylase protein, the rate limiting enzyme in dopamine synthesis, in the Mimetic poison frog (Ramitomeya imitator). We compared the knockdown efficacy of each morpholino at two different time points and the effect of knockdown on tadpole behavior. To quantify tyrosine hydroxylase knockdown efficiency, we developed a quick and inexpensive dot blotting method to evaluate protein levels within a single tadpole brain. We compared this method to chemiluminescent imaging and found both methods are sufficient to assess protein levels and discuss important differences in blot visualization techniques. Finally, we found morpholino knockdown reduced tadpole swimming, consistent with other studies implicanting dopamine as important in motor behaviors. We complement this protocol with a discussion of common challenges, suggestions for troubleshooting, and ideas for future improvement. Extending morpholino delivery and protein knockdown assessment to diverse amphibian species and labs with low budgets will enable the field to move forward more rapidly in the study of tadpole physiology and behavior.
Guidelines
These parameters are a suggested starting point. Optimizing the protocol for species-specific applications to maximize transfection efficiency is recommended.
Materials
Injection, Electroporation, and in vivo Imaging Materials
Ethyl 3-Aminobenzoate Methanesulfonate (Millipore Sigma Catalog #10521)
Sodium Bicarbonate (Millipore Sigma Catalog #S6014)
Josh's Frogs R/O Rx
Parafilm M Sealing Film (Millipore Sigma Catalog #HS234526B)
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 (MFI Medical 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)
Nanoject II Variable Volume Automatic Injector (Drummond Scientific Catalog #3-000-204)
GeneTools Standard and/or Vivo MOs designed against target of interest
Nuclease-Free Water (Millipore Sigma Catalog #W4502)
Kimwipes (Fisher Scientific Catalog #06-666)
Standard Disposable Transfer Pipettes (Fisher Scientific Catalog #13-711-7M)
Micro Detail Paint Brush
Stereoscope with a GFP filter
Dot Blot Materials
Tris-HCl (JT Baker Catalog #4103-01)
NaCl (Millipore Sigma Catalog #S9888)
10x SDS Stock (Invitrogen Catalog #24730020)
Tween-20 (Biotum Catalog #22002)
Forceps (Fisher Scientific Catalog #12-000-157)
Curved forceps (Fine Science Tools Catalog #91117-10)
Fine scissors (Fine Science Tools Catalog #91460-11)
Distilled water
cOmplete Mini EDTA-free table (Millipore Sigma Catalog #04693159001)
Motorized Tissue Grinder (Fisherbrand Pellet Pestle Cordless Motor Catalog #12-141-361)
Reusable or Disposable Pellet Pestles (Fisherbrand RNAse-Free Disposable Pellet Pestles Catalog #12-1411364)
Invitrogen Qubit 4 Fluorometer Catalog #Q33238
Centrifuge
1.5 mL Eppendorf Tubes (Fisher Scientific Catalog #02682003)
Nitrocellulose Membrane (Thermo Scientific Catalog #88018)
5% Nonfat Dry Blotting Grade Dry Milk (Bio-Rad Catalog #1706404)
Bovine Serum Albumin (Millipore Sigma Catalog #A3059-100G)
Primary Antibody for target of interest (Millipore Sigma Anti-Tyrosine Hydroxylase MAB318)
Primary Antibody for housekeeping reference protein (Abcam Anti-GAPDH AB181602)
HRP-Conjugated Secondary Antibody (Abcam Goat Anti-Mouse Secondary with HRP AB6789)
ECL Visualization Kit (Bio-Rad Catalog #1705060)
Chemiluminescent Imaging System (Azure Biosystems Catalog #76501-636)
Opti-4CN Substrate Kit (Bio-Rad, Hercules, CA, ca# 1708235)
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 of tadpole water
5m
Store at 4C for up to one week
Electroporation Set-Up
Electroporation Set-Up
8m
8m
Remove the tips from two 1 mL serological pipettes using scissors and fasten them together with electrical tape and/or hot glue
Figure 1. Two serological pipettes with electrode needles (see Step 4) attached together using electrical tape and hot glue to secure
5m
Mount the pipettes onto the micromanipulator and run one needle electrode wire through each pipette. Ensure the electrode needles are 1 mm apart and parallel to one another
FIgure 2. Two serological pipettes with electrode needles secured onto micromanipulator mount
1m
Attach the wires from the electrode needles to the capacitor and then to the stimulator. Set the stimulator parameters to 1 pps, 15 ms duration, 1 ms delay, and 30-50 V
Figure 3. Top view of electrode needles, capacitor, and stimulator connected
Figure 4. Side view of electrode needles, capacitor, and stimulator connected
Note
These parameters are a suggested starting point — optimizing the protocol for target molecules of interest and species-specific applications is recommended
2m
Injection Set-Up
Injection Set-Up
45m
45m
Reconstitute morpholino solution (0.5 - 1.0 mM working concentration) in nuclease-free water
3m
Pull micropipettes from glass capillaries using a pipette puller
10m
Backfill the micropipette with mineral oil using a 28 gauge needle and 1 mL syringe
5m
Using forceps, break the micropipette at an angle to create a beveled tip. Performing this task using a dissection microscope is recommended
5m
Place the micropipette onto the injector plunger and tighten the collet
3m
Select an injection volume between 23-56 nL and set the injection rate to slow
Figure 5. Nanoject II settings for injection volumes
Figure 6. Example injection setting at 23.0 nl and slow
Note
This injection volume range is a suggested starting point. Morpholinos can be slightly toxic, so optimizing the appropriate injection volume for species-specific application is recommended.
1m
Empty enough mineral oil to remove any bubbles and load 1-2 uL of morpholino solution
5m
Pipette 1-2 uL of morpholino solution onto a piece of Parafilm
1m
Gently dip the needle into the morpholino solution on the Parafilm and fill the micropipette without introducing air bubbles
2m
Construct a platform out of clay molded into the shape of a hill and fix on top of an empty Petri dish(es)
Figure 7. Hill-shaped bed mounted on top of Petri dishes to hold tadpoles
5m
Place the platform under a dissection microscope with the electrode on one side and the injector on the other
Figure 8. Full injection and electroporation set-up with clay tadpole platform positioned under dissection microscope
5m
Injection and Electroporation
Injection and Electroporation
22m
22m
Anesthetize the tadpole by placing it in a Petri dish of room temperature 0.03% MS-222 for 3-5 minutes
5m
Confirm the tadpole is completely sedated by checking for movement in response to stimuli
1m
Cover the clay platform with a Kimwipe damp with tadpole water
1m
Transfer the tadpole to the clay platform with a cut transfer pipette
Figure 9. Anesthetized tadpole laying dorsal side up on top of Kimwipe moistened with frog water on the clay platform
1m
Adjust the position of the tadpole to be dorsal side up with its head in the clay depression
1m
Orient the tadpole so the head is facing away from the injector
1m
Lower the injector and insert the pipette needle into the brain, targeting the brain ventricle
5m
Inject the morpholino solution
Note
We recommend injecting with 23-56 nL (detailed in Step 11) to start for species-specific optimization
2m
Remove the pipette from the tadpole brain. Allow 10-20 sec before electroporation
Note
If delivering standard morpholinos, continue with the protocol. If delivering Vivo MOs, skip to step 30
1m
Orient the tadpole so the head is facing away from the electrode
1m
Lower the electrode needles until it is in full contact with the tadpole head on either side of the injection site
2m
Deliver the electrical pulses (4 total, half regular polarity and half reverse polarity)
Figure 10. Close-up of stimulator settings with pulse switch and polarity switch circled in red
Note
The given pulse number is a suggested starting point. Optimizing the protocol for targets of interest and species-specific applications is recommended
1m
Deliver 2 pulses on regular polarity with a 1s interval between each pulse
Switch the polarity to reverse
Deliver 2 pulses on reverse polarity with a 1s interval between each pulse
Remove the electrode from the tadpole skin
Transfer the tadpole to fresh tadpole water for several hours to recover
In vivo Visualization of Standard MO
In vivo Visualization of Standard MO
1h 15m
1h 15m
At least 24 hrs after electroporation, anesthetize the tadpole by placing it in a Petri dish of room temperature 0.03% MS-222 for 3-5 minutes
Note
Fluorescent signal can be imaged as soon as 24 hours post-electroporation. However, knockdown is most effective after previously translated protein of interest has been degraded. Optimizing timing based on target turnover rate and species-specific application is recommended
5m
Move the tadpole to a new, small, and empty Petri dish using a cut transfer pipette
1m
Place the Petri dish underneath the fluorescent stereomicroscope
1m
Turn on the stereomicroscope and set the filter to GFP
1m
Open the imaging software
1m
Locate the tadpole under the stereomicroscope and zoom in, centered on the brain area
3m
Capture and save the fluorescent image
3m
Transfer the tadpole to fresh tadpole water for several hours to recover
1h
Preparing Protein Extraction and Dot Blot Solutions
Preparing Protein Extraction and Dot Blot Solutions
30m
30m
Prepare 1x Tris Buffered Saline (TBS)
15m
Mix 800mL distilled water with 6.05g Tris HCl and 8.76g NaCl
10m
Adjust pH to 7.6
4m
Add distilled water to bring to volume
1m
Store at 4C for up to 3 months
Prepare 2% SDS in 1x TBS
5m
Mix 800 uL of 1x TBS with 200 uL of 10x SDS
Store at 4C for up to 3 months
Prepare 7x protease inhibitor stock
5m
Add 1 cOmplete Mini EDTA-free tablet to 1.5 mL of distilled water
Store at 4C for up to one month
Prepare lysis buffer
5m
Mix 857 uL 2% SDS in 1x TBS with 143 uL 7x protease inhibitor stock
Prepare 1x TBST wash buffer
5m
Mix 500 mL of 1x TBS with 500 uL of Tween-20 (=0.01%)
Protein Extraction
Protein Extraction
2h
2h
Anesthetize the tadpole by placing it in a Petri dish of room temperature 0.03% MS-222 for 5 minutes
5m
Sacrifice by rapid decapitation and dissect out tadpole brain
10m
Put the brain directly into 50-100uL of lysis buffer in a clean 1.5 mL microcentrifuge tube
Note
For later lysis and processing, rapid freeze with dry ice and store at -80C
1m
Homogenize the brain by hand with pestle or motorized tissue grinder
4m
Centrifuge tubes at 13000 rpm/18928 rcf for 90 min
1h 30m
Measure protein concentration on Qubit
Note
Samples can be stored at -20 C before continuing the protocol. If proceeding from -20 C storage, allow samples to thaw on ice before proceeding with the dot blot
30m
Calculate sample amounts for desired protein concentration (i.e. 15 ug protein) brought to volume in 1x TBS for dotting 6 ul of sample onto each of two nitrocellulose membranes
30m
Transfer the supernatant to a new 1.5 mL microcentrifuge tube
10m
Dot Blot
Dot Blot
1d 1h 30m
1d 1h 30m
Using a narrow-mouth pipette tip, carefully dot 2-4 uL of samples onto two nitrocellulose membranes in the same pattern
30m
Allow the membranes to fully dry, then rewet in 1x TBST for 10 min
30m
Block non-specific binding in 5% dry milk in 1x TBST for 1 hour at room temperature with gentle agitation
1h
Incubate with primary antibody against target of interest on one membrane and primary antibody against reference protein on second membrane (1:100-1000) in 5% dry milk in 1x TBST for 1 hour at room temperature with gentle agitation
Note
We recommend optimizing primary antibody concentrations using manufacturer’s recommendation and pilot dot blots before data collection
1h
Wash three times with 1x TBST for 10 min each at room temperature with gentle agitation
30m
Incubate with secondary antibody with HRP conjugate (1:2000-5000) with 2% BSA in 1x TBST for 1 hour at room temperature with gentle agitation
1h
Wash three times with 1x TBST for 5 min each at room temperature with gentle agitation
Note
If chemiluminescent imaging only is desired, perform steps 59, 60 and 64. If brightfield imaging only is desired, skip to step 61. For both imaging techniques, perform the rest of the protocol as written.
15m
Incubate with Clarity ECL substrate (equal volumes of each component, mixed) for 5 min in dark
5m
Image the membranes using a chemiluminescent imaging system (i.e. ChemiDoc)
Note
We recommend trying different lengths of exposure to minimize background. If subsequent brightfield imaging is desired, we recommend waiting overnight to perform the colorimetric reaction.
10m
Combine DI water, Opti-4CN dilutant, and Opti-4CN substrate according to Opti-4CN development kit and incubate membranes overnight
18h
Rinse membranes in DI water for up to 10 min. Let dry at RT for up to 30 min and seal with tape
45m
Image on brightfield next to normalized step ladder
15m
Measure optical density in ImageJ using mean gray values for each sample blotted across both membranes
1h
Calculate an optical density ratio of your protein of interested to that of your reference housekeeping protein
30m