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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.

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In utero Electroporation of Mouse Embryo Brains
小鼠宫内胚胎脑皮层的DNA电转移

神经科学 > 发育 > 形态建成
作者: Xuecai Ge
Xuecai GeAffiliation 1: Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology (MIT), Cambridge, USA
Affiliation 2: , Howard Hughes Medical Institute, Cambridge, USA
For correspondence: xuecaige@stanford.edu
Bio-protocol author page: a46
Vol 2, Iss 14, 7/20/2012, 10435 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.231

[Abstract] 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)

[Abstract] 这是一种无侵入性的将基因转入发育的大脑的转基因技术。在这个方法中,DNA被注射入胚胎脑的侧脑室,通过电穿孔的方法被吸收到细胞中。然后,胚胎细胞在体内正常条件继续发育。可以在固定的时间点检测转入的目的基因的在子宫电穿孔中的影响。这种基因允许精确的下调或过表达目的基因。来自于其他基因的补偿性作用很少发生;同时也能避免可能的慢性的有害影响。

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)

Equipment

  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.

Procedure

  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

Recipes

  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.

Acknowledgments

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

References

  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.

材料与试剂

 

1.        EndoFree的质粒提取试剂盒(QIAGEN, 货号#12362

2.        固绿成纤维细胞趋化因子(Sigma-Aldrich,货号# F7252

3.        无菌盐水(0.9% 氯化钠)

 

设备

 

1.        微量吸管(硼硅酸盐Borosilicate with filament O.D.: 1mm, I.D.: 0.78 mm,10cm长)(Sutter 仪器,货号#BF100-78-10

2.        微量吸管拆卸器P-97/ IVFSutter仪器,诺瓦托,CA

3.        电转化仪(Electro-Square Porator CUY21, NEPA Gene)

4.        铂板镊子类型的电极(国际Protech , 货号#CUY650-P5)

5.        环钳 (Fine Science Tools公司, 货号#11101-09)

6.        齿状钳(Fine Science Tools公司,货号# 11000-12)

7.        精细手术剪 (Fine Science Tools公司,货号# 14060-09)

8.        测针夹持器 (Fine Science Tools公司, 货号#12003-15)

9.        Silk Black Braided Suture黑丝编织物缝合 (Ethicon Inc公司, 货号#K871)

10.     3”x3” 消毒纱布 (Dynarex公司,货号# 3353)

11.     方形脉冲电转仪 CUY21 (Nepagene公司, 日本). because during the surgery both of your hands are occupied to hold the animal and the electrodes, respectively.因为在外科手术中,两手要分别拿动物和电极,所以需要一个脚踏板。

12.     口部吸管 (Sigma公司,货号# A5177)

13.     光纤灯源尼康 MKII 或者其他。

14.     异氟醚麻醉药蒸馏器 (Porter仪器公司 ,型号 100-F)

强烈推荐用异氟醚作为麻醉药。 如果异氟醚不可用,那腹膜内注射氯胺酮(80-100 mg/kg)      与甲苯噻嗪 (5-10 mg/kg)的混合物也能达到同样的效果,但是避免使用三溴乙醇,因为其对晶胚有毒。

 

实验步骤

 

15.     准备好DNA注射使用的小型微量吸管

1)       用微量吸管的钳子将硼硅酸盐的小型微量吸管拉下。建议的相关参数的设定:压力, 500; heat热量, 800; pull拉力, 30; velocity速度, 40; 次数, 这个过程中应该产生一个带着管肩的逐渐变细的吸液管。

2)       用手术钳在距吸液管管肩处1.2 cm处切断拉下来的吸液管。

3)       用防水的荧光笔标记切下来的吸液管的顶端,以便能分清他们的底部末端。在吸液管上每个5mm长用防水的荧光笔做标记(每5mm的跨度对应5μl)。

 

16.     DNA的准备

1)       EndoFree的质粒试剂盒纯化质粒。所提的质粒DNA的终浓度应高于1ug/ulDNA的浓度越高产生的荧光越亮。

2)       DNA的溶液中加1/10体积的1%浓度的固绿作为一种失踪物(终浓度0.1%

17.     电穿孔

1)       设置电穿孔仪器。对于E15晶胚:35V,工作(开):50毫秒,休息(关):950毫秒,5脉冲。电压应该随着动物的年龄的增加而提高。我通常是每天1-2V的增加。

2)       设置口部吸液管:将一个拉下来的微型吸液管插入口吸液管中。往微型吸液管中抽取大约10 ulDNA溶液。

3)       用异氟醚麻醉一只时控的怀孕的小鼠(或者腹膜内注射氯胺酮甲苯噻嗪

4)       经过异氟醚的初步麻醉,将小鼠腹部朝上放在手术操作台上。然后从异氟醚蒸发器拿下面罩盖在小鼠的鼻子上。用实验室的胶带纸将小鼠的腿捆在手术操作台上。

5)       70%的乙醇清洗腹部。用小的剃刀(可以在Walgreen买到)剃去腹部的毛。用中间有个3cm长的细缝的折叠的纱布盖住腹部。(图A

6)       用无菌盐水将纱布变得潮湿。

7)       用精细手术剪纵向在皮肤中间上切开大约2cm长的切口。然后在腹腔的肌肉组织上划出一个大约1.5cm的切口。腹壁上的中间线(像根白线样能被看到)不应该因为要更好的康复而被切开。

8)       使用环钳通过收缩晶胚之间的间隙小心的取出子宫(但不是胎盘和胚胎)。注意别损伤到到胎盘或者与子宫连接的血管壁是很重要的。在整个外科手术中,你应该通过不断的加预热的(37°C)盐溶液。

9)       用齿形手术钳轻轻的夹好子宫,仔细认真的用环钳将一个胚胎细胞推入子宫壁中。通过纤维光学照明,子宫是透明的。胚胎前脑胞是清晰可见的。

10)   一手用环钳夹住胚胎,用另外一只手拿住口移液管。用小型微量吸管插入新大脑皮层2-3mm,这样吸管的顶端将正好在侧脑室(图B

11)   在脑室中注射1-3 μl DNA溶液。如果定向正确的话,侧面的脑室将会填满DNA固绿,且呈新月形的形状。

12)   用环钳夹住已注射DNA的胚胎,将其前后轴平行的通过子宫,然后将镊子状的铂板电极通过大脑部位,正极靠近注射的一边。向胚胎传送电脉冲:开(工作) 35 V, 毫秒, 关(休息)950毫秒, 5次脉冲。

13)   继续另一个胚胎。你可以注射一窝所有的晶胚除了那个离阴道最近的那个。避免那个能有助于避免流产。

14)   小心仔细的将晶胚放回腹腔中。

15)   用温暖的盐水填满腹腔。尽最大努力将晶胚放回初始的位置是很重要的。用PBS填满腹腔有助于帮助晶胚滑会他们最初的位置。

16)   用缝线关住子宫壁上的外科切口,然后缝合皮肤。

17)   将动物保持在37oC ,直到它从异氟醚中恢复过来。

 

 A

 

(来自于Saito and Nakatsuji, 2001, Dev Biol )

 B

 

 

 

(来自于Saito and Nakatsuji, 2001, Dev Biol )

试剂配方

 

1.         1%溶于水的Fast green(过滤室温保存)

2.         氯胺酮与甲苯噻嗪的混合物使用之前应正确配制

组合: 1 ml氯胺酮 (浓度100 mg/ml)

0.5 ml甲苯噻嗪 (浓度 20 mg/ml)

8.5 ml 无菌盐水或磷酸缓冲液.

剂量: 0.1 ml /10 gm体重

 

参考文献

 

1.        Ge X., Frank C.L., Calderon de Anda F., 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., Tsai L.H. (2009). Disrupted in schizophrenia 1 regulates neuronal progenitor proliferation via modulation of GSK3β/β-catenin signaling. Cell 136(6): 1017-31.

3.        Saito T., Nakatsuji N. (2001). Efficient gene transfer into the embryonic mouse brain using in vivo electroporation. Developmental Biology 240(1): 237-46.

 

 

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How to cite this protocol: Ge, X. (2012). In utero Electroporation of Mouse Embryo Brains. Bio-protocol 2(14): e231. DOI: 10.21769/BioProtoc.231; Full Text



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1/16/2014 3:09:23 PM  

Cardin Julie
IRCM

Hi,
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

1/17/2014 2:27:48 PM  

Xuecai Ge (Author)
Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology (MIT), USA


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.

Xuecai



3/4/2014 1:28:58 PM  

Cardin Julie
IRCM

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,
Julie

3/4/2014 1:31:22 PM  

Cardin Julie
IRCM

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.

3/17/2014 1:51:04 PM  

Xuecai Ge (Author)
Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology (MIT), USA


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.

Xuecai

4/28/2014 2:55:07 PM  

Cardin Julie
IRCM

Hi,
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,
Julie

4/28/2014 7:13:21 PM  

Xuecai Ge (Author)
Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology (MIT), USA

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.

Best,
Xuecai

4/29/2014 2:34:11 PM  

Cardin Julie
IRCM

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 7:55:12 PM  

Xuecai Ge (Author)
Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology (MIT), USA


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.

4/30/2014 12:36:27 PM  

Cardin Julie
IRCM

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

4/30/2014 12:41:13 PM  

Cardin Julie
IRCM

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!

5/5/2014 10:33:15 PM  

Xuecai Ge (Author)
Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology (MIT), USA

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.

-Xuecai



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