TITLE: Use and efficiency of morpholinos in Neotropical tadpole brains
AUTHORS: Sarah C. Ludington, Julie Butler, Chloe Golde, Lauren A O'Connell

[DESCRIPTION]
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.

[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 of tadpole water
SECTION: Anesthesia Preparation
2. Store at 4C for up to one week
SECTION: Injection Set-Up
6. Reconstitute morpholino solution (0.5 - 1.0 mM working concentration) in nuclease-free water
SECTION: Injection Set-Up
7. Pull micropipettes from glass capillaries using a pipette puller
SECTION: Injection Set-Up
8. Backfill the micropipette with mineral oil using a 28 gauge needle and 1 mL syringe
SECTION: Injection Set-Up
9. Using forceps, break the micropipette at an angle to create a beveled tip. Performing this task using a dissection microscope is recommended
SECTION: Injection Set-Up
10. Place the micropipette onto the injector plunger and tighten the collet
SECTION: Injection Set-Up
11. Select an injection volume between 23-56 nL and set the injection rate to slow
    
 



SECTION: Injection Set-Up
12. Empty enough mineral oil to remove any bubbles and load 1-2 uL of morpholino solution
SECTION: Injection Set-Up
13. Pipette 1-2 uL of morpholino solution onto a piece of Parafilm
SECTION: Injection Set-Up
14. Gently dip the needle into the morpholino solution on the Parafilm and fill the micropipette without introducing air bubbles
SECTION: Injection Set-Up
15. Construct a platform out of clay molded into the shape of a hill and fix on top of an empty Petri dish(es)

 

SECTION: Injection Set-Up
16. Place the platform under a dissection microscope with the electrode on one side and the injector on the other
  

SECTION: Electroporation Set-Up
3. Remove the tips from two 1 mL serological pipettes using scissors and fasten them together with electrical tape and/or hot glue 

SECTION: Electroporation Set-Up
4. 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
  

SECTION: Electroporation Set-Up
5. 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

    
 




SECTION: Injection and Electroporation
17. Anesthetize the tadpole by placing it in a Petri dish of room temperature 0.03% MS-222 for 3-5 minutes
SECTION: Injection and Electroporation
18. Confirm the tadpole is completely sedated by checking for movement in response to stimuli
SECTION: Injection and Electroporation
19. Cover the clay platform with a Kimwipe damp with tadpole water
SECTION: Injection and Electroporation
20. Transfer the tadpole to the clay platform with a cut transfer pipette
  

SECTION: Injection and Electroporation
21. Adjust the position of the tadpole to be dorsal side up with its head in the clay depression
SECTION: Injection and Electroporation
22. Orient the tadpole so the head is facing away from the injector
SECTION: Injection and Electroporation
23. Lower the injector and insert the pipette needle into the brain, targeting the brain ventricle
SECTION: Injection and Electroporation
24. Inject the morpholino solution
 

SECTION: Injection and Electroporation
25. Remove the pipette from the tadpole brain. Allow 10-20 sec before electroporation
 

SECTION: Injection and Electroporation
26. Orient the tadpole so the head is facing away from the electrode
SECTION: Injection and Electroporation
27. Lower the electrode needles until it is in full contact with the tadpole head on either side of the injection site
SECTION: Injection and Electroporation
28. Deliver the electrical pulses (4 total, half regular polarity and half reverse polarity)
  
 


SECTION: Injection and Electroporation
28.1. Deliver 2 pulses on regular polarity with a 1s interval between each pulse
SECTION: Injection and Electroporation
28.2. Switch the polarity to reverse
SECTION: Injection and Electroporation
28.3. Deliver 2 pulses on reverse polarity with a 1s interval between each pulse
SECTION: Injection and Electroporation
29. Remove the electrode from the tadpole skin
SECTION: Injection and Electroporation
30. Transfer the tadpole to fresh tadpole water for several hours to recover
SECTION: In vivo Visualization of Standard MO
31. 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
 

SECTION: In vivo Visualization of Standard MO
32. Move the tadpole to a new, small, and empty Petri dish using a cut transfer pipette
SECTION: In vivo Visualization of Standard MO
33. Place the Petri dish underneath the fluorescent stereomicroscope
SECTION: In vivo Visualization of Standard MO
34. Turn on the stereomicroscope and set the filter to GFP
SECTION: In vivo Visualization of Standard MO
35. Open the imaging software
SECTION: In vivo Visualization of Standard MO
36. Locate the tadpole under the stereomicroscope and zoom in, centered on the brain area
SECTION: In vivo Visualization of Standard MO
37. Capture and save the fluorescent image
SECTION: In vivo Visualization of Standard MO
38. Transfer the tadpole to fresh tadpole water for several hours to recover
SECTION: Protein Extraction
44. Anesthetize the tadpole by placing it in a Petri dish of room temperature 0.03% MS-222 for 5 minutes
SECTION: Protein Extraction
45. Sacrifice by rapid decapitation and dissect out tadpole brain
SECTION: Protein Extraction
46. Put the brain directly into 50-100uL of lysis buffer in a clean 1.5 mL microcentrifuge tube
 

SECTION: Protein Extraction
47. Homogenize the brain by hand with pestle or motorized tissue grinder
SECTION: Protein Extraction
48. Centrifuge tubes at 13000 rpm/18928 rcf for 90 min

51. Transfer the supernatant to a new 1.5 mL microcentrifuge tube
49. Measure protein concentration on Qubit
 

50. 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
SECTION: Dot Blot
52. Using a narrow-mouth pipette tip, carefully dot 2-4 uL of samples onto two nitrocellulose membranes in the same pattern 
SECTION: Dot Blot
53. Allow the membranes to fully dry, then rewet in 1x TBST for 10 min
SECTION: Dot Blot
54. Block non-specific binding in 5% dry milk in 1x TBST for 1 hour at room temperature with gentle agitation
SECTION: Dot Blot
55. 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
 

SECTION: Dot Blot
56. Wash three times with 1x TBST for 10 min each at room temperature with gentle agitation
SECTION: Dot Blot
57. 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
SECTION: Dot Blot
58. Wash three times with 1x TBST for 5 min each at room temperature with gentle agitation
 

SECTION: Dot Blot
59. Incubate with Clarity ECL substrate (equal volumes of each component, mixed) for 5 min in dark
SECTION: Dot Blot
60. Image the membranes using a chemiluminescent imaging system (i.e. ChemiDoc)
 

SECTION: Preparing Protein Extraction and Dot Blot Solutions
39. Prepare 1x Tris Buffered Saline (TBS)
SECTION: Preparing Protein Extraction and Dot Blot Solutions
39.1. Mix 800mL distilled water with 6.05g Tris HCl and 8.76g NaCl
SECTION: Preparing Protein Extraction and Dot Blot Solutions
39.2. Adjust pH to 7.6

SECTION: Preparing Protein Extraction and Dot Blot Solutions
41. Prepare 7x protease inhibitor stock
SECTION: Preparing Protein Extraction and Dot Blot Solutions
42. Prepare lysis buffer
SECTION: Preparing Protein Extraction and Dot Blot Solutions
40. Prepare 2% SDS in 1x TBS
SECTION: Preparing Protein Extraction and Dot Blot Solutions
39.3. Add distilled water to bring to volume
SECTION: Preparing Protein Extraction and Dot Blot Solutions
39.4. Store at 4C for up to 3 months
SECTION: Preparing Protein Extraction and Dot Blot Solutions
40.1. Mix 800 uL of 1x TBS with 200 uL of 10x SDS
SECTION: Preparing Protein Extraction and Dot Blot Solutions
40.2. Store at 4C for up to 3 months
SECTION: Preparing Protein Extraction and Dot Blot Solutions
41.1. Add 1 cOmplete Mini EDTA-free tablet to 1.5 mL of distilled water
SECTION: Preparing Protein Extraction and Dot Blot Solutions
41.2. Store at 4C for up to one month
SECTION: Preparing Protein Extraction and Dot Blot Solutions
43. Prepare 1x TBST wash buffer
SECTION: Preparing Protein Extraction and Dot Blot Solutions
42.1. Mix 857 uL 2% SDS in 1x TBS with 143 uL 7x protease inhibitor stock
SECTION: Preparing Protein Extraction and Dot Blot Solutions
43.1. Mix 500 mL of 1x TBS with 500 uL of Tween-20 (=0.01%)
SECTION: Dot Blot
64. Measure optical density in ImageJ using mean gray values for each sample blotted across both membranes
SECTION: Dot Blot
61. Combine DI water, Opti-4CN dilutant, and Opti-4CN substrate according to Opti-4CN development kit and incubate membranes overnight
SECTION: Dot Blot
62. Rinse membranes in DI water for up to 10 min. Let dry at RT for up to 30 min and seal with tape
SECTION: Dot Blot
63. Image on brightfield next to normalized step ladder
SECTION: Dot Blot
65. Calculate an optical density ratio of your protein of interested to that of your reference housekeeping protein