In Utero Electroporation Based Gene Delivery: A Technique to Inject Plasmid DNA into Embryonic Cerebellar Cells of Murine Embryos

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1. Perform In Utero Electroporation

1. Breed 6 to 8-week-old mice and prepare the tools for in utero electroporation. Cross male homozygous Rosa26-CAG-LSL-Cas9-P2A-EGFP mice with female CD-1 mice. Time the pregnancy of the CD-1 mice from the day the vaginal plug is detected (E0.5). Embryos are electroporated at day E13.5.
   1. Pull borosilicate glass capillaries (inside diameter: 0.6–0.8 mm) with a micropipette puller. Use the following settings: P = 500, Heat = 560, Pull = 150, Vel = 75, Time = 250. Label the capillary tip with black ink for better visualization during the injection. Trim the capillary taper under a stereomicroscope using a ruler and sharp needle tweezers and trim the tip at a length of about 6 mm.
   2. Maintain the unity of the capillaries to keep the experiment reproducible. Create a one-sided beveled edge during the trimming. The tip should be as sharp as possible for easy penetration of the uterine wall and to avoid tissue damage of the embryo.
      NOTE: Alternatively, use a micro-grinder to adjust a 35° angle. The capillaries can be stored at room temperature in a 10 cm Petri dish under pathogen-free conditions.
   3. Autoclave the surgical instruments.
   4. Mix the sgRNA-plasmids in equal parts with pT2K-IRES-Luc to a final concentration of at least 1 µg/µL for each plasmid and color the plasmid-solution with fast green (final concentration of 0.05%). Prepare 1% fast green stock solution with endotoxin-free distilled water and filter through a 0.22 µm filter to remove dye particles from the solution.
      NOTE: Co-transfection of pT2K IRES-Luc is optional, but allows for identification of viable electroporated animals after birth using bioluminescence imaging. Prepare a sufficient amount of plasmid-mix for the total number of pregnant mice to be operated. Use about 15–20 µL mix per mouse (~12 embryos). Proceed immediately with the surgery.
2. Prepare mice for surgery
   1. Anesthetize pregnant CD-1 mice with isoflurane. Induce anesthesia in a box with 3–4 vol-% isoflurane (oxygen flow rate: 1.5 L/min) and maintain it with 2 vol-% during surgery via nose cone.
      NOTE: The complete surgery should not take longer than 30 min. Reduce the number of injected and electroporated embryos, if necessary. Use sterile gloves for surgery.
   2. Place the mouse on its back on a heating pad and adjust anesthesia.
   3. Inject analgesic (Metamizol, 800 mg/kg body weight, subcutaneously (s.c.), 24 h depot).
      NOTE: You may use other authorized analgesics than Metamizol.
   4. Apply eye ointment to prevent eye-dryness during surgery.
   5. Fix limbs with tape and sterilize the abdomen with a disinfectant. Cover the mouse with gauze, leaving only the surgical area exposed. Spread 70% ethanol over surgical area and gauze.
   6. Make a skin incision of about 2 cm in length and widen the gap gently with straight surgical scissors. Locate the linea alba and make a slightly smaller second incision through the peritoneum using tissue scissors with blunt tip.
   7. Moisten the opened abdominal cavity with pre-warmed sterile 1x PBS.
   8. Carefully extract the uterine horns from the abdominal cavity using ring forceps. Pull gently by grabbing the uterine wall solely at the gap between the yolk sacs of two neighbouring embryos. Avoid any pressure on the embryo while pulling it through the incision. Enlarge the incision if necessary and take extra care to position the uterine horns onto the abdomen while not compressing blood flow through the uterine artery.
      NOTE: In some cases, it is advised to extract one uterine horn at a time and replace it before proceeding with the second uterine horn.
3. Injection and electroporation of plasmid DNAs
   1. Aspirate approximately 15 µL of colored plasmid-solution with the prepared glass capillary. Avoid any air-bubbles during this process.
      NOTE: The plasmid-solution can clog the tip easily if its concentration is high. Test the capillary by releasing some DNA right before the injection.
   2. Gently hold the embryo with ring forceps and locate the neck area.
      NOTE: If the embryo is in a position difficult to inject, skip it and go for the next embryo. Increased repositioning of the embryo may increase chances of damage to them.
   3. Slowly pierce with the tip of the previously prepared glass capillary into the fourth ventricle by penetrating through the dorsal hindbrain and subsequently inject about 1 µL of the plasmid-mix. Confirm that the dyed DNA is not leaked from the brain and is visible as a diamond shaped structure.
      NOTE: As an option, use 2.5-fold magnifying glasses for better visualization of the fourth ventricle and surrounding blood vessels. Deliver electric pulses immediately after injection to prevent diffusion of the plasmid-solution to the other ventricles.
   4. Apply electric square pulses (5 pulses, 32 mV, 50 ms-on, 950 ms-off) with forceps-like platinum tweezers (see Table of Materials). Place the electrodes laterally with the negative pole covering the ear and the positive pole positioned at the cerebellar primordium over the uterine wall. Use 5 mm-diameter electrode plates for effective electroporation of E13.5 embryos.
      NOTE: Increase the survival rate of pups by manipulating less than 2/3^rd of the total number of embryos per dam. Avoid embryos too close to the cervix. If manipulated, they might cause complications during labor and jeopardize the entire litter. If electrodes are too close to the embryo's heart, this may cause cardiac arrest.

자료

Name	Company	Catalog Number	Comments
Autoclave band	Kisker Biotech	150262
Electro Square Porator	BTX	ECM830
Endofree Maxi Kit	Qiagen	12362
Ethanol	Merck	107017
Eye ointment (Bepanthen)	Bayer	81552983
Fast Green	Merck	104022
Filter (0.22 µm)	Merck	F8148
Forceps straight	Fine Science Tools	91150-20
Gauze (X100 ES-pads 8f 10 x 10 cm)	Fisher Scientific	15387311
Glass Capillary with Filament	Narishige	GD1-2
Heating Pad	ThermoLux	463265 / -67
Isoflurane	Zoetis	TU061219
Ring Forceps	Fine Science Tools	11103-09
Stereomicroscope	Nikon	C-PS
Surgical scissors	Fine Science Tools	91460-11
Surgical scissors with blunt tip	Fine Science Tools	14072-10
Tweezers w/5mm- disk electrodes Platinum	Xceltis  GmbH	CUY650P5
Metamizol	WDT
Non-sterile Silk Suture Thread (0.12 mm)	Fine Science Tools	18020-50
PBS (1x)	Life Technologies	14190169
Tissue scissors Blunt (11.5 cm)	Fine Science Tools	14072-10
pCAG-EGxxFP	Addgene	50716
pX330 plasmid	Addgene	42230