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Jan 2010

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In utero Electroporation of Mouse Embryo Brains    

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This is a non-invasive technique to introduce transgenes into developing brains. In this technique, DNA is injected into the lateral ventricle of the embryonic brains, and is incorporated into the cells through electroporation. Embryos then continue their development in normal conditions in vivo. The effects of genes of interest can be evaluated at certain time points after in utero electroporation. This technique allows acute knockdown or over expression of genes of interest. Compensatory effects from other genes are less likely to happen; it also circumvents possible chronic detrimental effects.

Keywords: In utero electroporation, Embryo, DNA, Neocortex, Brain

Materials and Reagents

  1. EndoFree Plasmid Kit (QIAGEN, catalog number: 12362 )
  2. Fast green FCF (Sigma-Aldrich, catalog number: F7252 )
  3. Sterile saline (0.9% sodium chloride)
  4. 70% ethanol
  5. Katemine + Xylzaine mixture (see Recipes)


  1. Micropipettes (Borosilicate with filament O.D.: 1mm, I.D.: 0.78 mm, 10 cm length) (Sutter Instruments, catalog number: BF100-78-10 )
  2. Micropipette puller P-97/ IVF (Sutter Instruments, Novato, CA)
  3. Electroporator (Electro-Square porator CUY21) (NEPA Gene)
  4. Platinum plate tweezers-type electrode (Protech International, model: CUY650-P5 )
  5. Ring forceps (Fine Science Tools, catalog number: 11101-09 )
  6. Serrated forceps (Fine Science Tools, catalog number: 11000-12 )
  7. Fine scissors (Fine Science Tools, catalog number: 14060-09 )
  8. Needle holder (Fine Science Tools, catalog number: 12003-15 )
  9. Silk Black Braided Suture (Ethicon, catalog number: K871 )
  10. 3”x3” Sterile Gauze (Dynarex, catalog number: 3353 )
  11. Square pulse electroporator CUY21 (Nepagene, Japan). A foot pedal is required, because during the surgery both of your hands are occupied to hold the animal and the electrodes, respectively.
  12. Mouth pipet (Sigma-Aldrich, catalog number: A5177 )
  13. Fiber optic light Nikon MKII or any other brand)
  14. Vaporizer for isoflurane anesthetic (Porter Instruments Company, model: 100-F )
    Isoflurane is highly recommended as anesthetics. If this is not available, intraperitoneal injection of the mixture of ketamine (80-100 mg/kg) and xylazine (5-10 mg/kg) will also work. But avoid avertin that is toxic to embryos.


  1. Preparation of micropipettes for DNA injection
    1. Pull the borosilicated micropeppets with the Micropipette puller. The following parameters are recommended: pressure, 500; heat, 800; pull, 30; velocity, 40; time, This program should produce a pipette with long shoulder that tapers gradually.
    2. Cut off pulled pipettes with forceps at ~1.2 cm from the shoulder of the pipettes.
    3. Mark tips of cut pipettes with a water-resistant magic marker in order to clarify their ends. Mark bodies of the pipettes every 5 mm length water-resistant magic marker (1 span with 5 mm corresponds to 5 μl).

  2. DNA preparation
    1. Purify plasmids using the EndoFree Plasmid Kit. The final concentration of the DNA should be higher than 1 μg μl-1. Higher concentrations of DNA produce brighter fluorescence.
    2. Add 1/10 volume of 1% fast green to DNA solution as a tracer (final 0.1%).

  3. Electroporation
    1. Set up the electroporator: For E15 embryos: 35 V, 50 mSec On, 950 mSec Off, 5 pulses. For younger or older animals, the voltage should decrease or increase accordingly. I usually use 1-2 V increment for each day.
    2. Set up the mouth pipette: Inset one pulled micropipette into the mouth pipette. Draw about 10 μl DNA solutions into the micropipette.
    3. Anesthetize a timed-pregnant mouse with isoflurane (or with i. p. injection of katemine + Xylazine).
    4. After initial anesthetization with isoflurane, put the mouse on the operation platform with the abdomen upside. Then fit the mask from the isoflurane vaporizer on the mouse nose. Tape the leg with lab tapes to the operation platform.
    5. Wash the abdomen with 70% ethanol. Shave the fur over the abdomen using a small razor (can be bought from Walgreen). Cover the abdomen with a piece of folded gauze which has a 3 cm long slit in its center (Figure 1).
    6. Damp the gauzer with sterile saline.
    7. Using the fine scissors cut the skin longitudinally for about 2 cm long at the midline. Then make an incision of about 1.5 cm in the muscle of the abdominal cavity. The midline (can be seen as a thin white line) of the abdominal wall should not be cut for well healing.
    8. With ring forceps, take out the uterus carefully by pinching gaps between embryos (but not either the placenta or embryos). It is important to take care not to damage either the placenta or the blood vessels connected with the uterus. Throughout surgery you should keep the uterus wet by applying prewarmed (37 °C) saline.
    9. Hold the utero gently with serrated forceps, and carefully push one embryo with the ring forceps to the uterine wall. By the illumination of the fiber optics, the uterin well is transparent. The telencephalon is clearly visible.
    10. Hold the embryo with the ring-forceps in one hand, and use the other hand to hold the mouth pipette. Penetrating the neocortex 2-3 mm with the micropipette, and the micropipette tip will be in the lateral ventricle (Figure 2).
    11. Inject 1-3 μl of the DNA solution into the ventricle. If correctly targeted, the lateral ventricle should be filled with DNA-fast green, and exhibits a crescent shape.
    12. Hold the DNA-injected embryo in parallel along its antero-posterior axis through the uterus with the ring forceps, and put the platinum plate tweezers-type electrode across the brain, with the “+” electrode next to the injected side. Deliver the electric pulses to the embryo: 35 V, 50 mSec On, 950 mSec Off, 5 pulses.
    13. Continue with another embryo. You can inject all the embryos in the litter except the one that is closest to the vagina. Avoiding that one will help to avoid abortion.
    14. Carefully put the embryos back to the abdominal cavity.
    15. Fill the cavity with warm saline. It is important to try your best to place the embryos into their original position. Filling the abdomen with PBS may help the embryos to slide back to their original position.
    16. Close the surgical incision in the uterine wall with suture, then suture the skin.
    17. Keep the animal warm at 37 °C until the recovery from anesthesia.

Representative data

Figure 1. Adapted from Saito and Nakatsuji, 2001, Dev Biol

Figure 2.Adapted from Saito and Nakatsuji, 2001, Dev Biol


  1. 1% Fast green in H2O (filtered and store at RT)
  2. Katemine + Xylzaine mixture should be made right before use:
    Combine: 1 ml Ketamine (concentration 100 mg/ml)
    0.5 ml Xylazine (concentration 20 mg/ml)
    8.5 ml sterile saline or PBS
    Dosage: 0.1 ml per 10 mg of body weight.


This protocol was adapted from Saito and Nakatsuji (2001), Ge et al. (2009) and Mao et al. (2010).


  1. Ge, X., Frank, C. L., Calderon de Anda, F. and Tsai, L. H. (2010). Hook3 interacts with PCM1 to regulate pericentriolar material assembly and the timing of neurogenesis. Neuron 65(2): 191-203.
  2. Mao, Y., Ge, X., Frank, C. L., Madison, J. M., Koehler, A. N., Doud, M. K., Tassa, C., Berry, E. M., Soda, T., Singh, K. K., Biechele, T., Petryshen, T. L., Moon, R. T., Haggarty, S. J. and Tsai, L. H. (2009). Disrupted in schizophrenia 1 regulates neuronal progenitor proliferation via modulation of GSK3beta/beta-catenin signaling. Cell 136(6): 1017-1031.
  3. Saito, T. and Nakatsuji, N. (2001). Efficient gene transfer into the embryonic mouse brain using in vivo electroporation. Dev Biol 240(1): 237-246.
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Ge, X. (2012). In utero Electroporation of Mouse Embryo Brains. Bio-protocol 2(14): e231. DOI: 10.21769/BioProtoc.231.

If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.

If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.

Wing Lam So
University of Hong Kong
Hi Xuecai
I am new to IUE technique and I tried quite many trials following your protocol, using E14.5-E16.5 C57BL/6J mice and giving te pulse with 25V, 980msec off, 5 pulses, trying to target the prefrontal cortex. However, either the mothers loses 4-5g weight 1-day post-surgery (which probably indicate the death of embryos), or the embryos will die in P2-3. I am really frustrated and hope if you can give me some advice:
1) Do you think holding the embryo too tight might lead to such severe high mortality rate? I usually try to hold it using a cotton swab as one of my finger to help me push the embryo towards the uterine wall. I usually hold it as tight as when I insert the micropipette towards embryos head, I will use some force such that the insertion would not push the embryo away from uterine wall. Am I right or did I still enforce too much pressure on embryos?
2) I usually will rotate the embryos with cotton swabs to a position that I can see their head more clearly, do you rotate the embryos? I am thinking if the rotation itself might disrupt the embryos and kill them.
3) I usually will leave the embryos locating at the edge of the uterine horn not receiving IUE. May I ask for your criteria to choose which embryos to do/not do IUE that ensure higher embryo/newborn survival rate?
3) As I saw from your protocol, your insertion is nearly perpendicular to the lateral ventricles. I also see others insert from the area near the eye and in elevated angle for about 35 degree, 20degree angle to the left from the brain midline. Did you try this angle too and what will you comment on these 2 different angles for insertion?

Thank you so much for your protocol and everything!
Best regards
M.Phil student
6/2/2020 4:24:34 AM Reply
Cardin Julie
1) Is there a relation between the efficiency for the plasmid gene expression with the voltage?
At high voltage I get more death, obviously.

2) Is there a relation beteween the efficiency for the plasmid gene expression with the concentration of the DNA plasmid injected?

Thank you very much,
Best regards
Julie Cardin
Research Assistant
1/16/2014 3:09:23 PM Reply
Xuecai Ge
University of California, Merced

Hi Julie,

Thanks for your questions.

1) Yes, there is a correlation between the transfection efficiency and the voltage. Higher voltage gives you higher transfection efficiency (larger areas transfected in the brain); but, as you mentioned, it also causes higher death rate. So you have to find the right balance. After many trials in my experiments, I finally got to 35V for E15, and increased 1-2V for E16, and used 40V for E17-E18. But after working with many of my colleagues, I found that the transfection efficiency and death rate can be affected slightly by how tight you hold the embryos with the electrodes. The tighter, the higher efficiency. Therefore the voltage I give here is a point to start with; you might have to adjust it based on your own experience.

2) Yes, higher DAN concentration is preferred. In my experiments, I used as high as possible. But too higher concentration makes the DNA solution viscous, and difficult to aspirate. The highest concentration I have used is 4ug/ul. Any concentration above 2ug/ul will give you satisfying results.

Hope this helps.


1/17/2014 2:27:48 PM Reply

Cardin Julie

Thanks a lot for your answers.

1) I am electroporating at 35V, 45V 60V and 80V many litters. I got some very nice results but it take me too many litters to get just one. Many embryos are born but most of them died at P2-P3 and I cannot find why? Any suggestion?

2) In which tissue do you use 35V at e15 ?

Thank you very much in advence.

Best regards,

3/4/2014 1:28:58 PM Reply

Cardin Julie

3) Do you use any drug after surgery to reduce inflammation and pain for the mother? I had been recommended to give a dose of Metcam. I also tried Carpofen but it seems that I got more death so I came back to Metcam.

3/4/2014 1:31:22 PM Reply

Xuecai Ge
University of California, Merced

Hi Julie,

In my experiments, I sac the animal before the pups are born. In this situation, I usually did not electroporate the entire litter, and made a map of which pups are electroporated. This helped the survival rate a lot. I did help with some colleagues who need to have the pups born. In this case, I would electroporate the entire litter, and we had similar problems as your had. The most possible reason is that the female did not take care of the pups well after birth, due to the trauma she has had. My suggestion would be to find a foster Mom for the new born pups, i.e. give the pups to another female who just delivered the baby. This needs to be done in a timely manner (within 1-2 day after the foster Mom deliver her baby), so that the foster mother will not kill the new baby. Another suggestion is to eletroporate a lot of pregnant females. This is what I did.

I electroporated the neocortex at E15, using 35V, exactly as what this protocol describes. In my hand, higher than 45V killed the pups.

I did use some pain killer to the females, as required by the animal facility in my university. I used Buprenox (Buprenorphine). I guess other brand with similar component will work. I injected into muscles, but not belly, to avoid further irritation to the pups.

Hope this helps.


3/17/2014 1:51:04 PM Reply

Cardin Julie

I always use pCAG as promotor for IUE in mice. Do you know if there is some other kind of promotor that work well in mice?
What about promotor GfalphaA,B,C , is it working well?

Thank you very much,

4/28/2014 2:55:07 PM Reply

Xuecai Ge
University of California, Merced

Hi Julie,

I have only tried CMV and CAG. CAG works better. I have not tried other promoters. Sorry that I cannot help with this question.


4/28/2014 7:13:21 PM Reply

Cardin Julie

I read in many papers that it is important to use endotoxin free Maxi prep kit to make your DNA for mice IUE. I always do it this way. But I wonder if it required to precipitate the DNA after to purify more or if it is already good after the Endotoxinfre Maxi prep?

Thank you

4/29/2014 2:34:11 PM Reply

Xuecai Ge
University of California, Merced

I used the DNA from endofree maxiprep as well. Never re-precipitate to purify further. I wold guess it won't make a big difference. DNA quality is important; but comparing to how gentle you treat your animal and what voltage you use, it is minor.

4/29/2014 7:55:12 PM Reply

Cardin Julie

Do you think it is better to resuspend your plasmid in TE, in water, PSB ...?

4/30/2014 12:36:27 PM Reply

Cardin Julie

When I was doing experiments in which I was electroporating at e12 and collecting embryos, around 66% of embryos survived. Now I need to keep the embryos electroporated until they are born P14. Most of them are born but many of them dies between P1 and P5.

Have you noticed a difference in survival when you want to keep animals older?

( I discussed with people who are doing chicks and they told me that they have a much higher mortality increase with age.)

Thank you!

4/30/2014 12:41:13 PM Reply

Xuecai Ge
University of California, Merced

Hi Julie,

Regarding the solvent for DNA: avoid TE (Tris-EDTA), because the EDTA is not good for the embryo. Use PBS because it has some salt and is conductive.

It is true that when you manipulate the late stage embryos, it will increase the survival rate. But then you have to think about which layer of neurons you want to label. Since the cortex is generated in an "inside-out" manner, Electroporating at E12 will most label deep layer neurons, and after E17 most labelled neurons will be at the superficial layer. It is not so absolute and there is usually a time window. Most people are interested in looking at Layer V neurons, and electroporating between E13-15 will be OK. But after E17, you won't label Layer V neurons, and the majority of them will be at layer II-III.


5/5/2014 10:33:15 PM Reply

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