基因传递到产后大鼠脑非心室质粒注射和电穿孔

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Summary

这个协议描述了一种非病毒的基因结构的交付方法的生活啮齿动物脑的某些区域。该方法由质粒制备,微管制造,新生鼠的小狗手术,显微注射的构造,并

Cite this Article

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Molotkov, D. A., Yukin, A. Y., Afzalov, R. A., Khiroug, L. S. Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation. J. Vis. Exp. (43), e2244, doi:10.3791/2244 (2010).

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Abstract

转基因动物的创作是一种标准的方法,在研究一个基因在体内的兴趣的功能。然而,许多基因敲除或转基因动物是没有生命力的,在这些修改后的基因表达或在整个有机体中删除的情况下。此外,各种补偿机制往往使人们难以解释的结果。补偿效果,无论是定时的基因表达或转染细胞的数量限制,可缓解。

产后非心室显微注射和体内电的方法,可以有针对性地提供基因,siRNA或染料分子直接到一个小区域的新生啮齿动物脑的兴趣。在传统的心室注射技术相比,这种方法允许非洄游类型的细胞转染。这里介绍的方法转染的动物,可用于双光子,例如,在活体成像对急性脑片电生理实验。

Protocol

1。简介

转基因动物的创作是一种强大的方法,在动物体1,解开疾病机制2,3以及操纵细胞的属性4的基因的功能进行调查。但是,该过程是相当费力,非常耗时且昂贵,因此有必要使用替代的基因传递方法,如病毒注射液5, 6和新生儿在子宫 7,8脑室注射。产后非室注射和电的方法,都有一套独特的优势:使用非病毒基因结构;创建一个本地的表达模式,在感兴趣的领域;非洄游类型的细胞原位转染的可能性,如皮质星形胶质细胞。

在这个视频中,我们展示了鸡β肌动蛋白启动子与CMV增强(pCAG EGFP) 9下使用的增强型绿色荧光蛋白的质粒编码的新生大鼠脑产后基因传递的详细程序。我们说明含有质粒注射液的准备,制造薄玻璃移液器和组装一个立体定向仪上的微量。然后,我们谈论anaesthetizing幼鼠与异氟醚,执行手术,大约在注射过程和有关使用的电钳电极,并在体内 porator。最后,我们简要地讨论了预期的结果,观点和这些实验过程中可能出现的困难。

2。质粒制备用于电穿孔

  1. 我们通常会准备10μL的质粒注射液200μL薄壁管。这个解决方案是足够的几个实验。
  2. 我们混合1μl的10X PBS(见材料和​​设备)和1μL0.1%的快速绿色水溶液(见材料和​​设备)8μL质粒的解决方案(见材料和​​设备)。
  3. 质粒在注射液中的终浓度应介于1和3μg/μL的。低浓度的质粒DNA会降低转染效率,而DNA浓度较高,将通过薄薄的玻璃毛细管尖端注射的粘性。

3。玻璃针准备

  1. 玻璃吸管的准备,我们使用的最大拉和供热参数灯丝硼硅玻璃毛细管(见材料和​​设备)和垂直电极的玻璃拉马(见材料和​​设备)。
  2. 然后,我们打破了使用一张纸的吸液管,实现了尖端10-20微米,直径的一角。
  3. 我们检查在显微镜下目标的尖端直径和形状。

4。微量组装

  1. 使用薄的聚合物毛细管(见材料和​​设备)填充矿物油10μL汉密尔顿注射器量的约1 / 3(见材料和​​设备)(见材料和​​设备)。避免气泡!
  2. 然后我们填补与矿物油的玻璃吸管和汉密尔顿注射器插入吸管。避免气泡!
  3. 然后固定在连接到控制器的微量注射器与玻璃吸管(见材料和​​设备)。
  4. 显微注射固定在立体定向仪(见材料和​​设备)的安装,使我们能够安全地沉浸到管与质粒注射液的玻璃吸管的尖端。
  5. 当针尖触及溶液的表面,浸它进一步的1或2毫米,并填写与注射用微量进样器控制器的混合约1μL移液器。

5。 Anaesthetizing动物

这里介绍的所有的程序进行,根据赫尔辛基法规大学动物实验。

  1. 对于每一个实验,我们填补约2毫升的异氟醚的气密注射器(见材料和​​设备)(见材料和​​设备)。
  2. 比注射器固定麻醉单位(见材料和​​设备)连接到空气的流动源,动物框和面罩固定在立体定向设置。
  3. 麻醉单元上,我们调整的气流,以约250毫升/分钟和4%的异氟醚的水平。
  4. 我们把小狗到2-5分钟的动物方块老鼠。
  5. 当小狗停止移动,放在加热垫(见材料和​​设备)连接的立体设置。
  6. 放入anaesthetizing面具的小狗的头喙部分。
  7. 它需要5到10分钟为小狗进入深麻醉。
  8. 检查一条尾巴捏麻醉深度和异氟醚流入减少约1.5%至2.0%。

6。外科,显微注射,电穿孔

  1. 治疗皮肤上的小狗的头与70%的乙醇。
  2. 使用小剪刀(见材料和​​设备)和薄钳(见材料和​​设备),切开皮肤从额头的小狗颈背。
  3. 侧身弯曲皮肤件,拉耳朵略成骷髅头和皮肤固定的听觉窍酒吧。
  4. 使用双目显微镜,我们找到的头骨的前囟点。
  5. 确定用立体坐标利益的地区。
  6. 上述本地区的注射针的位置和标记颅骨表面注射液下降到25-50 NL。
  7. 颅骨钻在标记点使用高速手术钻(见材料和​​设备),在显微镜下轻轻地,仔细地,直到液体出现在钻探面积。
  8. 修复与当前的导电凝胶头骨两侧electropotation钳电极(见材料和​​设备)(见材料和​​设备),以达到更好的导电性。
  9. 从钻的孔用小棉球取出液滴(见材料和​​设备),并根据注射部位的坐标的玻璃针DIP孔。
  10. 注入25至100 NL注射液在5至20 nL /秒的速度。
  11. 迅速取出吸管,electroporate立即。电穿孔技术是通过申请5个矩形脉冲持续时间50毫秒和振幅在频率为1 Hz,99 V。
  12. 取下电极和耳朵酒吧。
  13. 缝合切开的皮肤,使用一个哑(见材料和​​设备)和尖锐的镊子和手术线程(见材料和​​设备)的组合。
  14. 将温暖到15-30分钟,以帮助其麻醉后恢复室的小狗。
  15. 然后把小狗回到其母亲的笼子。

7。代表性的成果

在成功转染的细胞基因的表达会出现后约10小时的电,将保持稳定几个星期。

我们注射质粒陷入深深的皮质层或产后的第2天(P2),大鼠幼仔纹状体区,并在体内电穿孔进行如上所述。大脑切片厚度(400微米)被切断后2至6天(P4 - P8)在冷切片介质使用一个vibrotome(HEPES缓冲Earle的平衡盐溶液)10。切片在室温下的介质中使用蔡司Axioplan 2 LSM5帕斯卡尔共聚焦显微镜(德国蔡司)进行图像采集。

深皮质层和纹状体注射部位有许多转染的细胞表达EGFP,约200-300微米直径(图1A,B)在一个紧凑的区域位于。转染的细胞内,EGFP表达水平足够高,让薄细胞过程(图1C),包括与树突棘不同的形状(图1D)和轴突束(图1E)成像。强劲稳定的EGFP以及细胞后,电的可行性允许跟踪远端的轴突(如皮质终端(图1F)从细胞体的1.5-2毫米)。

图1
图1:(一)成功转染细胞在划定质粒注射部位的P4大鼠脑纹状体区域。 (二)转染的细胞在深皮质层P5大鼠脑皮层表面以下(约1000微米)。 (c)在转染细胞P8的大鼠脑纹状体区域。 (四)在P8的大鼠脑纹状体区域的转染细胞的树突状进程;箭头指示不同类型的树突棘和丝状伪足。 (五)转染细胞内抵押品的途径的轴突。 (六)轴突终止P8大鼠皮层(箭头)的软脑膜表面。比例尺是100微米,A,B,E和F,10微米C和D

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Discussion

到生活的啮齿类动物的大脑中的基因传递方法是建立在子宫电7,8,11,12和,最近, 产后电6。然而,这些方法的基础上脑室注射质粒DNA,这可能是多个应用程序的限制。例如,这些方法不容许针对某些脑区如海马,也不等非迁徙作为皮层星形胶质细胞细胞类型的转染的细胞。首先被用于基因运送到13小脑的神经元电耦合的非室注射。我们的协议说明非心室法对大鼠脑组织中的其他地区扩展。我们提出,我们的方法的方法是一种有用的替代病毒基因利益的禁区内交付产后大鼠组织5的方法。

尽管本方法的优点,可预期一些困难,讨论如下。

1.Optimal年龄的动物

在可能的情况下,年轻的新生幼鼠应使用,作为一种手段,以增加手术后转染的疗效和小狗的生存。在我们的手中,在P0幼崽太小,因为它是难以再现,在一定区域内使用大脑立体坐标从脑图谱可取得 14这个年龄段的目标。此外,在P0幼仔有可能妨碍缝制的皮肤薄嫩。 P3和P4是最方便的手术和立体定向操作相比,P0 - P2的动物,但他们有过敏异氟醚麻醉在手术过程中,可能会导致惊厥和呼吸中断。因此,最好的办法是在手术过程中,可预见的重复性microinjections足够大的P1 - P2的幼鼠。

用显微注射的2.Possible问题

当溶液(25-100 NL)小容量注射薄玻璃,尖脑组织施加反对压力,这是关键,小心避免气泡或泄漏。否则,以避免这些可能导致转染效率急剧下降。

立体坐标的3.Precision限制

尽管立体坐标精度,他们限制在0.3毫米的重复性15。在我们使用相同的年龄和体重的动物的经验,它有可能减少0.2-0.1毫米,这是足够CA1区海马区重现针对此限制。

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Acknowledgements

我们感谢配乐录制的视频,伊万Molotkov为CAG - EGFP质粒制备的三维动画和彼得Blaesse博士的帮助,叶卡捷琳娜Karelina。

这项工作是由来自芬兰的国际流动,芬兰文化基金会和芬兰科学院中心的赠款支持。

Materials

Name Type Company Catalog Number Comments
2A-sa dumb Tweezers, 115mm equipment XYtronic XY-2A-SA Treat with 70% ethanol for disinfection before use in surgical manipulations
Biological Temperature Controller with stainless steel heating pad equipment Supertech TMP-5b
Borosilicate tube with filament material Sutter Instrument Co. BF120-69-10 Glass needle
Disposable drills material Meisinger HP 310 104 001 001 008
Dulbeco’s PBS 10X reagent Sigma-Aldrich D1408
Dumont #5 forceps, 110 mm equipment Fine Science Tools 91150-20 Treat with 70% ethanol for disinfection before use in surgical manipulations
Ealing micr–lectrode puller equipment Ealing 50-2013 Vertical electrode glass puller
Ethilon monofil polyamide 6-0 FS-3 16 mm 3/8c material Johnson & Johnson EH7177H Surgical threads
Exmire micro syringe 10.0 ml equipment Exmire MS*GLLX00 Gas-tight syringe
Fast Green reagent Sigma-Aldrich F7252
Forceps electrodes equipment BEX LF650P3 Treat with 70% ethanol for disinfection prior to use
Foredom drill control equipment Foredom FM3545 Surgical drill power supply and control. Currently available analogue is micromotor kit K.1070 (Foredom)
Foredom micro motor handpiece equipment Foredom MH-145 Currently available analogue is micromotor kit K.1070 (Foredom)
Gas anesthesia platform for mice equipment Stoelting Co. 50264 Assembled on stereotaxic instrument
Isoflurane reagent Baxter Internationl Inc. FDG9623
Micro dressing forceps, 105 mm equipment Aesculap BD302R Treat with 70% ethanol for disinfection before use in surgical manipulations
Microfil material World Precision Instruments, Inc. MF34G-5 Micro syringe filling capillaries
Mineral oil reagent Sigma-Aldrich M8410
NanoFil Syringe 10 microliter equipment World Precision Instruments, Inc. NANOFIL Hamilton syringe
plasmid CAG-EGFP reagent Extracted and purified with EndoFree Plasmid Maxi Kit (Qiagen) and dissolved in nuclease free water to concentration 1.5 mg/ml
Pulse generator CUY21Vivo-SQ equipment BEX CUY21Vivo-SQ
Schiller electrode gel reagent Schiller AG 2.158000 Conductive gel
Small animal stereotaxic instrument equipment David Kopf Instruments 900
St–lting mouse and neonatal rat adaptor equipment Stoelting Co. 51625 Assembled on stereotaxic instrument.Treat earbars with 70% ethanol for disinfection before use in surgical manipulations
Student iris scissors, straight 11.5 cm equipment Fine Science Tools 91460-11 Treat with 70% ethanol for disinfection before use in surgical manipulations
Sugi absorbent swabs 17 x 8 mm material Kettenbach 31602 Surgical tampons
UMP3 microsyringe pump and Micro 4 microsyringe pump controller equipment World Precision Instruments, Inc. UMP3-1 Microinjector and controller
Univentor 400 Anesthesia Unit equipment Univentor 8323001

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References

  1. Gerlai, R., Clayton, N. S. Analyzing hippocampal function in transgenic mice: an ethological perspective. Trends Neurosci. 22, 47-51 (1999).
  2. McGowan, E., Eriksen, J., Hutton, M. A decade of modeling Alzheimer's disease in transgenic mice. Trends Genet. 2, 281-289 (2006).
  3. Cryan, J. F., Holmes, A. The ascent of mouse: advances in modeling human depression and anxiety. Nat. Rev. Drug Discov. 4, 775-790 (2005).
  4. Wells, T., Carter, D. A. Genetic engineering of neural function in transgenic rodents: towards a comprehensive strategy. J. Neurosci. Methods. 108, 111-130 (2001).
  5. Pilpel, N. reproducible transduction of select forebrain regions by targeted recombinant virus injection into the neonatal mouse brain. J. Neurosci. Methods. 182, 55-63 (2009).
  6. Boutin, C. Efficient in vivo electroporation of the postnatal rodent forebrain. PLoS One. 3, e1883-e1883 (2008).
  7. Saito, T., Nakatsuji, N. Efficient gene transfer into the embryonic mouse brain using in vivo electroporation. Dev. Biol. 240, 237-246 (2001).
  8. Saito, T. In vivo electroporation in the embryonic mouse central nervous system. Nat. Protoc. 1, 1552-1558 (2006).
  9. Matsuda, T., Cepko, C. L. Electroporation and RNA interference in the rodent retina in vivo and in vitro. Proc. Natl. Acad. Sci. U S A. 101, 16-22 (2004).
  10. De Simoni, A., Yu, L. M. Preparation of organotypic hippocampal slice cultures: interface method. Nat. Protoc. 1, 1439-1445 (2006).
  11. Walantus, W. In utero intraventricular injection and electroporation of E15 mouse embryos. J. Vis. Exp. (2007).
  12. Walantus, W., Elias, L., Kriegstein, A. In utero intraventricular injection and electroporation of E16 rat embryos. J. Vis. Exp. (2007).
  13. Umeshima, H., Hirano, T., Kengaku, M. Microtubule-based nuclear movement occurs independently of centrosome positioning in migrating neurons. Proc. Natl. Acad. Sci. U S A. 104, 16182-16187 (2007).
  14. Ashwell, K., Paxinos, G. Atlas of the Developing Rat Nervous System. 3rd edition, Academic Press. (2008).
  15. Paxinos, G., Watson, C. The Rat Brain in Stereotaxic Coordinates. 6th edition, Academic Press. (2007).

Comments

8 Comments

  1. Is it possible to use this technique with adult rats?

    Reply
    Posted by: mandres@bio.puc.cl m.
    September 22, 2010 - 5:47 PM
  2. We tried the method only for rat pups under the age of P8. Also we have found that transfection efficiency (or at least expression efficiency) decreased when we use P5-P8 pups.
    Another factor that should be kept in mind - recovery after electroporation. It means that the brain, as neuronal network, is a kind of electrical nework and if apply electrical shock on mature network consequences might be too critical.

    Reply
    Posted by: Anonymous
    September 27, 2010 - 9:17 AM
  3. How did you connect a glass pipette with the syringe? I bought WPI nanofil but its bore did not fit with 1.²mm glass pipette. Is there an adopter to connect them?

    Reply
    Posted by: Sho Y.
    October 13, 2010 - 3:12 AM
  4. We unscrewed a metal fixation cap from WPI's nanofil and also remove a rubber gasket. Then we reamed bores in cap and gasket with 1,3 mm drill and made a chamfer on the front side of the cap using ²,5 mm drill (like on original nanofil but bigger). Then the syringe was assembled. Regarding adaptors, I did not hear about such things for nanofil.

    Reply
    Posted by: Anonymous
    October 13, 2010 - 8:08 AM
  5. Thank you very much for detailed information.

    Reply
    Posted by: Sho Y.
    October 13, 2010 - 8:36 AM
  6. There are some new features for the method that we introduced after the paper was accepted and I think they will be very helpful:
    1. We now use 5% glycerol (in addition to all other listed components) in injection mix. That improves localization of transfected cells and prevents solution sticking to micropipette tip during manipulations.
    ². For better localization of transfected cells we leave the pipette inside the injection site after injection and during all electroporation cycles. Afterwards, the pipette must be removed slowly.
    3. For labeling of drilling position on skull surface we use ²-3 nl of injection mix now, thus having a tiny dot allows more precise targeting.

    Reply
    Posted by: Anonymous
    October 13, 2010 - 8:09 AM
  7. Nice method! Do you think it can be implemented in mice?
    Also, what is the transfection efficiency? Is it reproducible?

    Reply
    Posted by: Anonymous
    April 8, 2011 - 11:24 AM
  8. Thank you for your interest and questions.
    I think it can be implemented in mice as well, you just need to mention that the field intensity should be around 100-1²0 V/cm.
    Transfection effeciency is not very high comparing with viral transduction: if you will inject 100 nl of plasmid (4-6 kb) in concentration 3 micrograms/microliter (total amount 300 ng of DNA) there will be 50-²00 transfected cells. Thus estimated transfection effeciency is around 1 cell per ² ng of DNA. Transfection effeciency is also dependent on brain area and promoter used.
    The method is reproducible if you use the same conditions all the time. My advice is to do firstly several pilot transfections to determine an optimal injection volume, speed and coordinates for your personal case, thus you can get better reproducibility for the method.

    Reply
    Posted by: Anonymous
    April 12, 2011 - 10:28 AM

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