Jan 2010



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 (子宫穿孔), Embryo (胚胎), DNA (DNA), Neocortex (新皮层), Brain (脑)

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.


这是将转基因引入发育中的大脑的非侵入性技术。 在该技术中,将DNA注入胚胎脑的侧脑室,并通过电穿孔掺入细胞中。 胚胎然后在正常条件下在体内继续其发育。 可以在子宫内电穿孔后的某些时间点评估感兴趣的基因的效应。 这种技术允许急性敲减或过表达感兴趣的基因。 其他基因的补偿效应不太可能发生; 它也绕过可能的慢性有害影响。

关键字:子宫穿孔, 胚胎, DNA, 新皮层, 脑


  1. EndoFree Plasmid Kit(QIAGEN,目录号:12362)
  2. 快速绿FCF(Sigma-Aldrich,目录号:F7252)
  3. 无菌盐水(0.9%氯化钠)
  4. 70%乙醇
  5. Katemine + Xylzaine混合物(参见配方)


  1. 微量移液器(Borosilicate,长丝O.D.:1mm,I.D .: 0.78mm,10cm长)(Sutter Instruments,目录号:BF100-78-10)
  2. 微量移液器P-97/IVF(Sutter Instruments,Novato,CA)
  3. 电穿孔仪( Electro-Square porator CUY21 )(NEPA Gene)
  4. 铂板镊子型电极(Protech International,型号:CUY650-P5)
  5. 环形钳(Fine Science Tools,目录号:11101-09)
  6. 锯齿钳(Fine Science Tools,目录号:11000-12)
  7. 精细剪刀(Fine Science Tools,目录号:14060-09)
  8. 针架(Fine Science Tools,目录号:12003-15)
  9. Silk Black Braided Suture(Ethicon,目录号:K871)
  10. 3"×3"无菌纱布(Dynarex,目录号:3353)
  11. 方形脉冲电穿孔仪CUY21(Nepagene,Japan)。 需要一个脚踏板,因为在手术过程中,两只手都被分别握住动物和电极
  12. 口移液管(Sigma-Aldrich,目录号:A5177)
  13. 光纤灯尼康MKII或任何其他品牌)
  14. 用于异氟烷麻醉的蒸发器(Porter Instruments Company,型号:100-F) 强烈建议使用异氟烷作为麻醉剂。 如果不可用,腹膜内注射氯胺酮(80-100mg/kg)和赛拉嗪(5-10mg/kg)的混合物也将起作用。 但避免对胚胎有毒的avertin


  1. 制备用于DNA注射的微量移液管
    1. 用Micropipette拉出器拉动硼硅化的微型地毯。 推荐以下参数:压力,500; 热,800; 拉,30; 速度,40; 时间,这个程序应该生产一个长肩逐渐渐变的移液器。
    2. 用距离移液管肩部约1.2厘米的镊子切下吸取的移液管。
    3. 标记吸管的提示,用防水魔术标记,以澄清他们的结束。 标记吸管的主体每5毫米长度防水魔术标记(1跨度与5毫米对应于5微升)。

  2. DNA制备
    1. 使用EndoFree质粒试剂盒纯化质粒。 DNA的最终浓度应高于1μg/μl -1。 更高浓度的DNA产生更亮的荧光
    2. 向DNA溶液中加入1/10体积的1%快速绿色作为示踪剂(最终0.1%)

  3. 电穿孔
    1. 设置电穿孔:对于E15胚胎:35 V,50 mSec开,950 mSec关,5脉冲。 对于年龄较大或较大的动物,电压应相应降低或增加。 我通常每天使用1-2伏增量。
    2. 设置嘴移液器:插入一个微孔吸入嘴吸管。 将约10μlDNA溶液吸入微量移液管
    3. 麻醉一个定时怀孕的老鼠用异氟烷(或用i。p。注射katemine +赛拉嗪)。
    4. 用异氟烷初次麻醉后,将小鼠放在手术台上腹部上方。然后将来自异氟醚蒸发器的面罩贴在鼠标鼻子上。将带有实验室磁带的腿绑在操作平台上。
    5. 用70%乙醇洗腹部。使用一把小剃刀剃掉腹部的毛皮(可以从Walgreen购买)。用一块折叠的纱布覆盖腹部,在其中央有一个3厘米长的狭缝(图1)。
    6. 用无菌盐水冲洗纱布。
    7. 使用细剪在纵向切开皮肤约2厘米长在中线。然后在腹腔的肌肉中做出约1.5cm的切口。腹壁的中线(可以看作是一条细的白线)不应该被切割以良好愈合。
    8. 用环镊子,通过捏胚胎之间的间隙(但不是胎盘或胚胎)仔细取出子宫。重要的是注意不要损伤胎盘或与子宫相连的血管。在整个手术中,你应该通过应用预热(37℃)的盐水保持子宫湿。
    9. 用锯齿镊子轻轻握住子宫,仔细推动一个胚胎与环镊子到子宫壁。通过光纤的照射,子宫孔是透明的。端脑清晰可见。
    10. 用一只手握住环镊子的胚胎,用另一只手握住嘴移液器。用微量移液管穿透新皮层2-3毫米,微量吸头位于侧脑室(图2)。
    11. 注入1-3微升的DNA溶液到心室。如果正确靶向,侧脑室应填充DNA-快速绿色,并呈现新月形
    12. 用注射过的胚胎沿着前后轴平行穿过子宫,用环形镊子,并将铂板镊子型电极穿过大脑,"+"电极在注射侧旁边。给胚胎输送电脉冲:35 V,50 mSec On,950 mSec Off,5脉冲。
    13. 继续另一个胚胎。你可以将所有的胚胎注入窝里,除了最接近阴道的那个。避免这种情况有助于避免流产。
    14. 小心地将胚胎放回腹腔。
    15. 用温盐水填充空腔。 重要的是尽力将胚胎放置到原始位置。 用PBS填充腹部可以帮助胚胎滑回到它们的原始位置
    16. 用缝合线关闭子宫壁的手术切口,然后缝合皮肤
    17. 保持动物温度在37°C,直到从麻醉恢复


图1.改编自Saito和Nakatsuji,2001,Dev Biol

图2.来自Saito和Nakatsuji,2001, Dev Biol


  1. 1%H 2 O中的快速绿色(过滤并在RT存储)
  2. Katemine + Xylzaine混合物应在使用前立即进行:
    0.5ml甲苯噻嗪(浓度为20mg/ml) 8.5ml无菌盐水或PBS




  1. Ge,X.,Frank,C.L.,Calderon de Anda,F.and Tsai,L.H。(2010)。 Hook3与PCM1相互作用以调节近中心材料装配和神经发生的时间。 Neuron 65(2):191-203。
  2. Mao,Y.,Ge,X.,Frank,CL,Madison,JM,Koehler,AN,Doud,MK,Tassa,C.,Berry,EM,Soda,T.,Singh,KK,Biechele, ,TL,Moon,RT,Haggarty,SJ和Tsai,LH(2009)。 Cell 136(6):1017-1031。
  3. Saito,T。和Nakatsuji,N。(2001)。 使用体内电穿孔将高效基因转移到胚胎小鼠脑中。/a> Dev Biol 240(1):237-246
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Ge, X. (2012). In utero Electroporation of Mouse Embryo Brains. Bio-protocol 2(14): e231. DOI: 10.21769/BioProtoc.231.



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

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

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

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

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

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

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

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

Cardin Julie

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

4/30/2014 12:36:27 PM

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

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