TITLE: Bulk in vivo electroporation (single cell)
AUTHORS: Anya Suppermpool

[DESCRIPTION]
This protocol will allow you to express single (or near single/mosaic) cell expression in the tectum of larvae. This protocol is adapted from (Hoegler and Horne, 2010; Nikolaou et al., 2015). If you would like to do focal-electroporation (into cell) to trace projections/neurites please see (Henriques et al., 2019) or any older Bianco paper. 

Electroporation is a way to deliver plasmids or vectors into living embryos. For my experiments, I want to see larvae/neurons at 6-9dpf so I electroporate at 3dpf. But if you wish you do younger see Hoegler (24hpf) - likely that you need to adapt some things as younger larvae are curled and will be more fidget-y.

Note: depending on what you want to express/and how much of it, electroporation time may differ. For example, AS wants one-neuron expression which gets roughly 10-30% of electroporated fish (depending on expression see Inject and electroporate section). AS then wants say 15-20 positive embryos => meaning 150-200 embryos electroporated => taking roughly 13:00-17:30/18:00 (5hr) long. 


[STEPS]
SECTION: Equipments
1. Stimulator SD9 (Grass Instruments) - borrow from Isaac Bianco (Ask Isaac before use!)
SECTION: Equipments
2. Electroporation penTM
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3. Micro glass needle (0.58mm inside diameter, Sutter Instrument, Germany, BF100-58-15) pulled using a micropipette puller (Model P-87 Sutter Instrument, Germany) – normal injection needle 
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4. Petridish ( 100 mm × 20 mm/normal size) lid + to contain your embryos
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5. Glass slide
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6. Water Bath at 42C (to keep agarose warm)
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7. 25mL plastic pipette to mount fish (or can use glass pipette but because glass has smaller opening/diameter agarose tends to get stuck more quickly so need a glass of hot/warm water to store/de-agarose the glass pipette
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8. Forceps (fine) and scalpels (want straight edge/45degree not curved blade for precisions)
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9. Pipette tips microloader (for loading plasmid into needle AND orienting larvae)
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11. Dissection microscope
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10. Pressure injection system (microinjector) and needle manipulator
SECTION: Making Electroporation PenTM
20. Constructing electroporation electrodes pen   

SECTION: Making Electroporation PenTM
21. Use wire cutter to cut plastic cover off exposing wire (wire end of the electrode)
SECTION: Making Electroporation PenTM
22. Electrode should be about 1mm apart (to fit larvae head in between)
SECTION: Making Electroporation PenTM
23. Run electrodes+wires through shell of a BIC/BIRO pen (other brand are available)
SECTION: Making Electroporation PenTM
24. Cover the wire with shell of a BIC pen for egonomy 
SECTION: Making Electroporation PenTM
25. Secure/waterproof the gap between two electrodes and pen shell  using epoxy-resin and parafilm 
SECTION: Making Electroporation PenTM
26. Use heat-shrink tubing to hold electrode wires together as well as connect between wires and pen shell.
SECTION: Reagents
12. 
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13. Fish water and tricaine 25x (communal fridge)
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14. Low-melting agarose in fish water (methylene blue-free)
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14.1. For electroporation use low so 1-1.5%
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14.2. For imaging (live) AS use 2-3%
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14.3. AS: use agarose in glass bottles not eppendorf tubes as this holds heat better, holds more + plus can be stored in water baths to keep temperature. 
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15. DNA plasmid of interest and/ or transgenic line (if using Gal4/UAS system) 
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15.1. AS electroporate Gal4 (FoxP2.A:Gal4FF) into Tg(UAS:FingR.PSD95)* stable line. AS only did this way as UAS:FingR lines (due to negative feedback mechanisms needed to be 1: in a stable line/genomically integrated, 2: ideally in higher copies). But you can do whichever in theory that scientifically suits your experiment. E.g. Nikolaou et al co-electroporated Gal4 and UAS:GCamP. 
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16. DNA plasmid (>500ng/uL, best at 1ug/uL)
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17. Tol2 transposase mRNA (>150ng/uL)** 
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17.1. tol2 transposase mRNA was in vitro transcribed from the NotI-linearized pCS-TP6287 plasmid (plasmid# 1151 Wilson lab database, UCL) using an SP6 mMESSAGE mMACHINE Kit (Ambion, USA). RNA was purified using RNA Clean & Concentrator Kits (Zymo Research, USA).
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18. Electroporation buffer (180 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 5 mM HEPES, pH 7.2)
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18.1. AS make this in 2X and dilute fresh everytime pre-electroporation (with fish water tricaine to make 0.02% Tricaine (1X, MS-222, Sigma-Aldrich)
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19. Phenol red (0.03%) 
SECTION: How to mount fish for electroporation
27. Pre mounting, screen your larvae to have the correct UAS/Gal4 background
SECTION: How to mount fish for electroporation
27.1. AS: incross UAS:FingR so screen for the strongest expressing FingR+ larvae (background Gal4). As found that stronger expression yields higher electroporated fish (30%) vs regardless of expression level (10%).
SECTION: How to mount fish for electroporation
27.2. AS: Electroporate at 3dpf.
SECTION: How to mount fish for electroporation
28. Tricaine (1X) larvae that you would like to mount. One batch at a time as mounting/injecting-electroporating/unmounting may take a while each round. AS typically do two plates(lids) at once and each lid containing 8 larvae (16 larvae per round in total) the whole round takes about ~25 minutes. 
SECTION: How to mount fish for electroporation
29. When larvae are definitely anesthetized mount larvae (one at a time onto the lid with glass slide with the agarose). Note: onto the plastic lid and not onto the glass slide. Glass slide only there to align the larvae.
SECTION: How to mount fish for electroporation
29.1. How AS mount: *make sure* agarose is warm enough (if not you will boil the embryo). To do this, AS put agarose onto back of AS’ hand to check temperature + if using glass pipette putting pipette into agarose bottle, pipette should NOT steam up if the correct temperature. 
SECTION: How to mount fish for electroporation
29.2. Pipette up larvae using glass/plastic pipette with as little water as possible and place larvae into glass bottle with agarose.
SECTION: How to mount fish for electroporation
29.3. Pick that larvae up using pipette and drop a little droplet of agarose-containing larva onto the surface of petridish lid (open side up). 
SECTION: How to mount fish for electroporation
29.4. Quickly and gently use trimmed microloader to orient the larva dorsal up. 
SECTION: How to mount fish for electroporation
29.5. Repeat this for the rest of your larvae on your lid (AS recommend 10-12 larvae)
SECTION: How to mount fish for electroporation
30. Now you have (say 5) larvae in droplets of agarose on your dish lid. Once they have solidified (but not too long that larvae hypoxic and gets ill so within 3mins max as there’s other steps after this) you can use your trimmed microloader to move the larva-droplet into position along and perpendicular to glass slide (facing away) (See Fig 3)  

SECTION: How to mount fish for electroporation
31. Pour agarose along these droplet into a line, mobilizing larvae to the glass slide and glass slide to the dish lid. 
SECTION: How to mount fish for electroporation
32. Once agarose has solidify, fill the half of the petridish lid with larvae with electroporation buffer (with 1X traicaine) 
SECTION: How to mount fish for electroporation
33. Cut agarose along larvae (both) side of body (Figure) and also in front of larvae head for needle and electrode to access (as much as you can) 
SECTION: How to mount fish for electroporation
34. Repeat this for the rest of your larvae/dish
SECTION: Inject and electroporate
35. Stimulator settings: Duration: 5ms, Power: 85V, Frequency: 200Hz, Delay:0
SECTION: Inject and electroporate
36. Prepare solution and load into injection needle.
SECTION: Inject and electroporate
36.1. AS in needle mix: FoxP2.A:Gal4FF (500ng/uL) + Tol2 mRNA (10-30ng/uL) + phenol red (0.03%) to help see solution in needle
SECTION: Inject and electroporate
37. Calibrate needle to inject 1nL using graticule ruler
SECTION: Inject and electroporate
38. Inject solution into larvae’ midbrain ventricle (AS do 5-8nL).
SECTION: Inject and electroporate
38.1. You should see the ventricle distends slightly   

SECTION: Inject and electroporate
38.2. Guess the more you inject the more chance of expression but AS found that 8nL max as larvae can get edema.
SECTION: Inject and electroporate
39. Electroporate immediately!
SECTION: Inject and electroporate
39.1. Five 5ms trains of 85 V voltage pulses at 200 Hz were
SECTION: Inject and electroporate
39.2. DNA solutions begins to diffuse immediately to other areas of the larvae, so electroporate as soon as possible (within 5-10seconds) post injection. Electroporate after injection of each embryo.
SECTION: Inject and electroporate
39.3. Obviously take out the needle from the ventricle before electroporate
SECTION: Inject and electroporate
40. Position electroporator penTM such that the positive electrode (red) is lateral and slightly dorsal to the hemisphere of the optic tectum targeted.
SECTION: Inject and electroporate
40.1. orientation of the electric field used here will specifically target only one hemisphere (right or left) of the developing optic tectum
SECTION: Inject and electroporate
40.2. AS: although because AS adapted using Tol2 mRNA, AS also sometimes see expression on the other hemisphere. ** so maybe don’t use Tol2 if you want to restricted to one side
SECTION: Inject and electroporate
41. Obviously *DO NOT TOUCH* embryo with electrode. This will harm/kill them. 
SECTION: Inject and electroporate
42. Repeat injection/electroporation to next larvae 
SECTION: How to unmount fish
43. Once done with all your larvae. Pour out electroporation buffer and replace with normal fish water. Wait 10-15mins for fish to recover 
SECTION: How to unmount fish
44. Free embryo from agarose by using forceps to gently hold agarose along the body (Do not touch the actual embryo) and scalpel to cut agarose from the head. Larvae should swim out freely.
SECTION: Screen for your result
45. Screen under table top microscope (if your expression is high enough). AS has to screen under confocal microscope to see single cells