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Organotypic Explants of the Embryonic Rodent Hippocampus: An Accessible System for Transgenesis
啮齿动物胚胎海马器官型外植体:一个可进行转基因的系统   

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参见作者原研究论文

本实验方案简略版
Proceedings of the National Academy of Sciences of the United States of America
Jul 2011

Abstract

This protocol describes the technique of ex-vivo electroporation to target embryonic hippocampal progenitors in an organotypic slice preparation. This technique allows gene perturbation for examining developmental processes in the embryonic hippocampus while retaining the environment and connectivity of the cells. Gene perturbation can include Cre-mediated recombination, RNAi-mediated knockdown, gene overexpression, or a combination of any of these. Ex-vivo electroporation can be performed at a wide range of embryonic stages, giving temporal control to the experimenter. Spatial control can be achieved more easily by preparing the brain in a Petri dish to target particular regions of the hippocampus. The electroporated explant cultures provide a highly tractable system for the study of developmental processes that include progenitor proliferation, migration and cell fate acquisition.

Keywords: Mouse hippocampus (小鼠海马), Embryo (胚胎), Electroporation (电穿孔), Hippocampal slice (海马切片), Organotypic explant (器官型外植体)

Background

The hippocampus presents a challenge in terms of accessibility due to its location in the caudomedial telencephalon. The embryonic hippocampus is even more inaccessible, requiring in utero surgical methods for experimental manipulation. Organotypic slice cultures circumvent this problem and at the same time retain many aspects of hippocampal field cytoarchitectonics, including molecular features and connectivity. While there are protocols that describe postnatal culturing of hippocampal explants from rodent brains (Stoppini et al., 1991; Opitz-Araya and Barria, 2011) these do not include genetic manipulation of the cells. The preparation of organotypic explants from the embryonic mouse hippocampus was first described in Tole et al. (1997). We extended this protocol by introducing ex-vivo electroporation of the embryonic brain prior to preparing the organotypic slices. Electroporation of the intact brain after introducing DNA into the telencephalic ventricle ensures that cells residing in and near the ventricular zone are targeted, and therefore provides an excellent means of inducing transgenesis in hippocampal progenitors. Data using this protocol were published in Subramanian et al. (2011). Here, we present detailed step-wise instructions including experimental ‘dos and don’ts’, and also illustrate key steps using photographs and movies, to aid new researchers in setting up this useful procedure.
Some advantages and applications of this protocol are:
1) Temporal control can be achieved by isolating the embryonic hippocampus at the desired stage to access early, mid, or late-gestation progenitors.
2) Spatial control can be achieved by orienting the electrodes to target the hippocampus or different areas of the cortex.
3) Cre-mediated recombination can be employed by electroporating Cre-GFP into embryos carrying the desired floxed alleles.
4) Overexpression constructs can be electroporated.
5) Embryonic lethal strains can be accessed by performing the procedure in the window of viability, and then further development can proceed in the organotypic explant.

Materials and Reagents

  1. Plastic Pasteur pipettes:
    3 ml (P-3) and 2 ml (P-1) (Taurus Biomedical)
    1.5 ml pipettes (RPI, catalog number: 147500 )
    Note: Henceforth these will all be referred to as ‘pipettes’. Procuring the correct size of pipette and cutting the shaft to the correct aperture size (see Figure 1) is key to being able to manipulate brains, hemispheres and explants without damaging them.


    Figure 1. Plastic Pasteur pipettes. A. Different sizes of pipettes used in this procedure: #1 (1.5 ml pipette), #2 (2 ml pipette), #3 (3 ml pipette) prepared by cutting #4 as shown. The cut is made just at the point where the pipette shaft diameter begins to taper, approximately 2 cm from the tip. B. Beaker with pipettes being sterilized in alcohol.

  2. 35 mm sterile Petri dishes (Laxbro, catalog number: PD-35 TC )
  3. Cell culture 6-well tissue culture plate (Thermo Fisher Scientific, catalog number: 140675 )
  4. Micropipette tip
  5. Glass Capillaries: Thin wall borosilicate tubing without filament (Sutter Instruments, catalog number: B100-75-10 )
  6. 100 mm Petri dishes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 150464 )
  7. Sterile blade
  8. Cell Culture Inserts: 0.4 µm (Millicell, Merck, catalog number: PICM03050 )
  9. Aspirator assembly (Sigma-Aldrich, catalog number: A5177-5EA )
  10. 50 ml syringe filter unit with 0.22 µm filter (Millex-GP, Merck, catalog number: SLGP033RS ) for sterilizing the culture medium
  11. 2 Squirt bottles (Tarsons, catalog number: 561100 ) (containing 70% and 100% ethanol)
  12. pCS2-EGFP plasmid (or any desired plasmid)
  13. Pregnant Swiss mice (SWR/J) (obtained from Tata Institute of Fundamental Research breeding facility)
  14. Absolute ethanol (Merck, catalog number: 107017 )
  15. Leibovitz’s L-15 medium (Thermo Fisher Scientific, GibcoTM, catalog number: 41300039 )
  16. Fast green dye (Sigma-Aldrich, catalog number: F7252 )
  17. Penicillin-streptomycin (P/S) (10,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  18. Dulbecco’s modified Eagle medium (Thermo Fisher Scientific, GibcoTM,catalog number: 12800017 )
  19. B27TM supplement (50x), serum-free (Thermo Fisher Scientific, GibcoTM, catalog number: 17504044 )
  20. Working solution of DMEM medium (see Recipes)

Equipment

  1. Horizontal flow hood (Kirloskar, Envair Electrodyne, catalog number: KCH-B )
  2. Glass beaker (Borosil, catalog number: 1000D21 )
  3. Tissue chopper (McIlwain, model: MTC/2E )
  4. Stereo microscope (Olympus, catalog number: SZ61 ) placed in a horizontal flow tissue culture hood
  5. Cell culture incubator (Thermo Fisher Scientific, model: HeracellTM 150 )
  6. Fine tools for manipulating embryonic brains:
    1. Dumont # 5 Forceps, Dumostar (Roboz Surgical Instrument, catalog number: RS-4978 )
    2. Dumont # 55, Forceps, Dumostar (Roboz Surgical Instrument, catalog number: RS-4984 )
    3. Micro-dissecting Spring scissors (Roboz Surgical Instrument, catalog number: RS-5603 )
  7. Coarse forceps (Roboz Surgical Instrument, catalog number: RS-5040 ) for handling the chopper stage disc
  8. Fine scissors (Fine Science Tools, catalog number: 14060-11 ) and tissue forceps (Roboz Surgical Instrument, catalog number: RS-8166 ) for opening the dam and removing the uterus
  9. Electrodes (3 mm, BTX, Harvard Apparatus, catalog number: 450487 )
  10. Electroporator (BTX, Harvard Apparatus, model: ECM 830 )
  11. Dual-stage glass micropipette puller (NARISHIGE, model: PC-10 )

Procedure

Ethics statement: All procedures followed the Tata Institute of Fundamental Research Institutional Animal Ethics Committee guidelines.

  1. Set-up (pre-preparation)
    Note: All steps after harvesting the embryos are performed in a horizontal flow hood.
    1. Wipe the surface of the tissue culture hood with 70% ethanol.
    2. Pour 70% ethanol into a 200 ml glass beaker and sterilize pipettes #1, #2, and #3 by aspirating the ethanol all the way into the bulb and then ejecting it out. Allow the residual ethanol to drain.
    3. Wash the pipettes with L-15 medium to remove traces of ethanol and keep standing in a sterile empty beaker near the dissection microscope for easy access.
    4. Place dissection instruments in a plastic tray containing 70% ethanol for 10 min, and then dry before using. Place in sterile tray for easy access during the procedure.
    5. Keep approximately six 35 mm Petri dishes filled with L-15 on ice.
    6. Add 1.25 ml of DMEM medium in the required number of wells of a 6-well tissue culture plate. Using a sterile forceps place one sterile cell culture insert per well. Transfer the 6-well plate to a 37 °C incubator to prewarm the medium. Typically, 5-8 organotypic explants can be accommodated on one cell culture insert depending on the embryonic stage.

  2. Preparation for injection
    1. Prepare 2 µg/µl of pCS2-EGFP plasmid (or any desired plasmid) in nuclease-free water. Lightly touch some fast green dye with the micropipette tip and introduce it into the DNA solution and mix. This enables visualization of the DNA solution during and after injection.
    2. For preparation of glass capillaries, set the capillary puller to one-step pull at a temperature of 62.5 °C.
    3. Pull capillaries using the puller and place them in a Petri dish on a capillary holder.
    4. Fit the capillary to a mouth aspirating rubber pipette and aspirate approximately 5-10 µl of the DNA solution containing fast green dye. This will last for approximately 2-3 ex-vivo injections.

  3. Harvesting embryos
    1. Sacrifice a pregnant dam of the desired stage (E13.5-18.5) using the procedures permitted by your Institute. The example shown below is E15.5 from a Swiss mouse. Nfia mutant embryos which were on the C57BL/6J background have also been used successfully in Subramanian et al., 2011.
    2. Wipe the abdominal skin with ethanol and make a longitudinal incision. Clean the incised area with 70% ethanol (squirt bottle) and using a new pair of sterile forceps and scissors make an incision in the abdominal wall.
    3. Lift out each arm of the uterus one by one taking care that it does not come in contact with the skin or hair of the dam. Cut the uterus at the lowest point and transfer to a dish containing sterile PBS. All procedures from this point onwards will take place inside the tissue culture hood.
    4. Using a dissection microscope, remove the embryos from the uterus carefully and place them in a sterile 100 mm Petri dish containing sterile 1x PBS.
    5. Decapitate the embryos and transfer the heads to a fresh sterile Petri dish containing L-15.
    6. Dissect out the brains and transfer using pipette #3 to a 35 mm dish containing L-15 kept on ice. After removing the brain, sever the hindbrain and midbrain, retaining only the forebrain including the olfactory bulbs. The presence of the olfactory bulb helps to position the telencephalic hemisphere in the correct orientation on the tissue chopper plate. The meninges should be retained and removed only after electroporation.
    Note: The multiple transfers (Steps C4-C6) to fresh Petri dishes containing sterile PBS/L-15 help to wash off any possible contamination and maintain sterility.

  4. Electroporation and organotypic slice preparation
    A summary cartoon illustrating the overall procedure is shown in Figure 2, together with a sample organotypic explant at the end of the culture period.


    Figure 2. Summary schematic of ex-vivo electroporation and organotypic slice preparation. A. Injection of DNA and electroporation of the intact brain; B. Sections containing electroporated progenitors (green circles); C. After 6 d in vitro, the explants display GFP-expressing pyramidal neurons (green triangles) residing within the pyramidal cell layer (yellow). These neurons initially extend axons (green) which grow towards the developing fimbria (arrowhead) and then encircle the periphery of the explant, presumably because they cannot exit the fimbria onto the filter. D. An image of an organotypic explant displaying an axon bundle growing into the fimbria and then around the explant (arrowheads). Scale bar = 100 µm. This figure and legend are modified from Figure 5, Subramanian et al., PNAS, 2011.

    1. Set the tissue chopper to 250 µm and insert a sterile blade onto the blade. Tighten the blade. Flush 100% ethanol over the blade using a squirt bottle and let the set-up dry.
    2. Wipe the paddle electrodes with ethanol and connect them to the electroporator.
    3. Set the parameters to the following: Voltage 50 V; Time 50 msec; Number of pulses 5; Pulse interval 999 msec.
    4. Inject the ventricle of the hemisphere to be electroporated with the desired DNA solution (mixed with Fast green), as shown in Figure 3A. The ventricles are hollow, and can be identified by their darker appearance, and the decrease in resistance when the micropipette punctures the neuroepithelium and enters the ventricle. It is recommended to perform unilateral injection and electroporation, however, the dye-DNA solution may sometimes diffuse into the connected contralateral ventricle (see Figure 3B). This is normal and does not affect the experimental setup. Fill the lateral ventricle with the solution until the dye reveals that the ventricular cavity is filled (see Figure 3B). Approximately 5-10 µl of solution can be injected into the ventricles depending on the age of the embryo used. Since the DNA is injected into the ventricles, the cells which have the best access to the DNA are the ventricular zone cells, which consist of dividing neuronal progenitors. This is the main population that is electroporated (Saito, 2006).


      Figure 3. Injection of DNA solution into telencephalic hemisphere and electroporation. A. Glass capillary (arrowhead) injecting DNA into the ventricular cavity. B. Paddle electrodes (positive and negative) positioned at an angle suitable for electroporating the hippocampus. (*In Figure 3B, the insulation of the negative electrode is chipped at the tip, so a portion of the paddle appears silver. This does not affect the electroporation.) Scale bar = 500 µm.

    5. Perform the electroporation immediately so that the DNA solution does not diffuse out of the ventricle. Place the electrodes at the angle shown in Figure 3B, such that the positive electrode is positioned on the contralateral side and the negative electrode is positioned on the ipsilateral side of the hemisphere to be electroporated. Since DNA is negatively charged, it will move towards the positive electrode and therefore display maximal electroporation on the medial side of the hemisphere.
    6. Initiate the pulse sequence on the electroporator. Successful passage of current is indicated by bubbles under the paddles around the region of electroporation. More bubbles are usually seen under the (-) paddle electrode.
    7. Transfer the electroporated brain to a 35 mm Petri dish containing ice-cold L-15 medium. Keep on ice for 5 min. In the meantime, electroporate other brains as per the experimental requirement. Transfer each brain to the ice-cold L-15.
    8. Once all the required brains are electroporated, separate the electroporated hemisphere and remove the meninges carefully.
    9. Place the hemisphere on the tissue chopper such that the medial face is in contact with the white disc on the stage, and the lateral (neocortex) faces up. Using pipette #1 gently remove excess medium. Excess medium will cause the hemisphere to wobble during the next step (chopping). While siphoning off the excess medium, orient the hemisphere’s rostro-caudal axis orthogonal to the blade so that coronal sections will be obtained (the olfactory bulb needs to point either right or left, not towards or away from you as in Figure 4).


      Figure 4. Placement of the dissected electroporated hemisphere onto the stage of the tissue chopper. Note that the spring clips that hold the chopping stage disc in place should be oriented in the R-L direction to prevent them from interfering with the blade. The telencephalic hemisphere orientation should be aligned taking this into account (Olfactory bulb pointing R or L). 

    10. Initiate chopping (Videos 1 and 2). Immediately, remove the disk holding the chopped tissue using two pairs of strong forceps. Flush the tissue into a clean L-15 containing dish containing cold L-15 (Video 3).

      Video 1. Chopping of electroporated hemisphere (viewed from the front)

      Video 2. Chopping of electroporated hemisphere (viewed from the side)

      Video 3. Flushing chopped tissue into a Petri dish

    11. The hemisphere will look like a loaf of sliced bread (Figure 5A). Turn the hemisphere over, so that the medial side faces up. Prod gently to release finger-like protrusions from the medial side that are individual slices of the hippocampus. Use the fine forceps to sever the hippocampus from the neocortex (dotted lines, Figure 5A). Collect hippocampal slices in a fresh 35 mm Petri dish containing ice cold L-15 medium.


      Figure 5. Isolating organotypic hippocampal slices from the chopped hemisphere. A. Image of a freshly chopped hemisphere displaying ‘sliced bread’ appearance. B. A single hippocampal explant isolated from the chopped hemisphere. C. The same explant in (B) re-oriented and labeled. CA1, CA3, hippocampal fields. DG, dentate gyrus. Scale bar = 500 µm.

    12. Remove the 6-well dish containing the cell culture inserts from the incubator and place under the dissection microscope (Figure 6).


      Figure 6. 6-well tissue culture dish with cell culture insert

    13. Using pipette #2 take up 5-6 hippocampal explants with some L-15 medium. Place the explants onto the filter disc one by one by gently applying pressure on the bulb of the pipette so that only one explant is dispensed, each in its own droplet of L-15 medium (Figure 7A). Use pipette #1 to gently aspirate out excess medium and use the fluid flow generated by the aspiration to gently position the explants so they do not touch each other and also do not stick to the walls of the cell culture insert (Figure 7B). There should be enough medium to keep explants from drying out but excess medium should be avoided to prevent explants from folding or floating away (Figures 7C and 7D). The explants will not dry; they can access the DMEM medium in the well through the filter.


      Figure 7. Placing hippocampal explants onto culture insert. A. The explants are placed in individual droplets of medium onto the cell culture insert using pipette #2. B. The excess fluid is removed using pipette #1. During this step, two explants that may have been deposited in the same droplet can be separated (C). D. Images of the explants when the procedure is complete.

    14. Incubate the hippocampal explants at 37 °C, 5% CO2. Change the medium every alternate day.
    15. Within several hours, the electroporation can be visualized if a fluorescent reporter construct was used. Within the next 3-5 days, the labeled progenitors differentiate into pyramidal neurons, extending apical processes towards the center of the curled explant, and axons course around the explant to the region of the fimbria (Figure 8).


      Figure 8. A single organotypic hippocampal explant imaged on consecutive days in vitro. A. After 1 day in vitro (DIV), electroporated cells are seen close to the ventricular zone (yellow dotted line). Apical processes are seen to extend toward the center of the explant arrowheads). (B) and (C) with additional days in vitro, cells begin to migrate away from the ventricular zone (yellow arrows) as they do in vivo, and form the pyramidal cell layer. D. By 6 days in vitro, the electroporated cells extend axons (white arrows) that can be seen encircling the explant. Images in (A-D) are composites consisting of multiple frames stitched together. Scale bar = 100 µm.

Data analysis

This ex-vivo electroporated hippocampal organotypic preparation can be used to assess whether neurons or glia are generated from progenitors. The presence of GFP-expressing axons in the fimbria is indicative of neurogenesis. See Subramanian et al. (2011), Figure 5, for images of explants electroporated with neurogenic or gliogenic constructs. These explants can also be immunostained for cell type-specific markers and analyzed for the distribution of excitatory/inhibitory neurons or glia as required.

Notes

  1. Reproducibility and variability: The extent of electroporation is variable, but can be made more reproducible with practice. The key issues to pay attention to are the angle of the electrodes and how carefully one holds the brain i.e., the contact of the tissue with the paddle electrodes. The brain needs to be completely submerged in the medium during the electroporation.
  2. Technical tips and cautionary points:
    1. The speed of producing the explants is critical to explant health. Keeping the tissue on ice-cold L-15 when it is not under the microscope or chopping stage is critical.
    2. Being able to identify ‘good’ hippocampal finger-like protrusions in a chopped brain also comes with practice. Typically, easy-to-recognize hippocampal morphology is seen at the caudal end of the chopped brain, whereas explants prepared from more medial locations are recognizable only by a tiny fimbria. A good rule of thumb is that if it doesn’t have a fimbria, it’s probably not a hippocampal explant, but a curved piece of neocortex masquerading as a hippocampus.
    3. The cell culture inserts fit best in a 6-well plate. Do not try to use individual 35 mm dishes–they don’t have the requisite height. The dish will not close properly, so the medium will dry or become contaminated.

Recipes

  1. Working solution of DMEM medium (50 ml)
    B27: 500 µl
    Penicillin-streptomycin: 500 µl of a 100x stock
    DMEM: 49 ml
    Filter sterilize using a 0.22 µm filter and store at 4 °C. Use this solution up to 1 week as suggested in the document manual for B27

Acknowledgments

We thank Leora D’Souza for assistance with the videography, Dr. S. Suryavanshi and the TIFR animal breeding facility for excellent support. This protocol was developed in the Tole lab using support from a Wellcome Trust Senior Fellowship (056684/Z/99/Z), a Swarnajayanti Fellowship (4/3/2005-SF), grants from the Department of Biotechnology, Government of India and intramural funds from TIFR-DAE to ST; a Kanwal Rekhi Career Development Award from the TIFR Endowment Fund to LS. ST is a recipient of the Shanti Swarup Bhatnagar award (Council of Scientific and Industrial Research, Government of India) and the Infosys Prize for Life Sciences (Infosys Science Foundation).
This protocol was used in Subramanian et al. (2011) Proc Natl Acad Sci U S A 108(27): E265-274. (Current address: Eli & Edythe Broad Center of Regeneration Medicine & Stem Cell Research, University of California San Francisco, CA 94143, USA.)
The authors have no conflicts of interest or competing interests.

References

  1. Opitz-Araya, X. and Barria, A. (2011). Organotypic hippocampal slice cultures. J Vis Exp (48).
  2. Saito, T. (2006). In vivo electroporation in the embryonic mouse central nervous system. Nature Protoc 1(3): 1552–1558.
  3. Stoppini, L., Buchs, P. A. and Muller, D. (1991). A simple method for organotypic cultures of nervous tissue. J Neurosci Methods 37(2): 173-182.
  4. Subramanian, L., Sarkar, A., Shetty, A. S., Muralidharan, B., Padmanabhan, H., Piper, M., Monuki, E. S., Bach, I., Gronostajski, R. M., Richards, L. J. and Tole, S. (2011). Transcription factor Lhx2 is necessary and sufficient to suppress astrogliogenesis and promote neurogenesis in the developing hippocampus. Proc Natl Acad Sci U S A 108(27): E265-274.
  5. Tole, S., Christian, C. and Grove, E. A. (1997). Early specification and autonomous development of cortical fields in the mouse hippocampus. Development 124(24): 4959-4970.

简介

该协议描述了在器官切片制备中靶向胚胎海马祖细胞的体外电穿孔技术。 该技术允许基因摄动检查胚胎海马体的发育过程,同时保持细胞的环境和连接性。 基因扰动可以包括Cre介导的重组,RNAi介导的敲低,基因过表达或这些中任何的组合。 可以在广泛的胚胎阶段进行体外电穿孔,为实验者提供时间控制。 空间控制可以通过在皮氏培养皿中准备大脑来瞄准海马的特定区域来实现。 电穿孔的外植体培养物提供了用于研究包括祖细胞增殖,迁移和细胞命运获取在内的发育过程的高度易处理的系统。

【背景】由于其位于尾端的端脑,海马在可达性方面提出了挑战。胚胎海马甚至更难以接近,需要用子宫外科手术方法进行实验操作。器官切片文化规避了这个问题,同时保留了海马场细胞构筑学的许多方面,包括分子特征和连接性。尽管有描述来自啮齿类动物大脑的海马外植体产后培养的规程(Stoppini等人,1991; Opitz-Araya和Barria,2011),但这些规程不包括对细胞的遗传操纵。首先在Tole等人(1997)中描述了从胚胎小鼠海马体制备器官外植体。我们通过在制备器官切片之前引入胚胎脑的体外电穿孔来扩展该协议。在将DNA导入远端脑室后,完整脑的电穿孔确保靶向存在于和附近心室区的细胞,并因此提供诱导海马祖细胞中转基因的极好手段。使用该协议的数据发表在Subramanian等人的(2011)中。在这里,我们提供详细的逐步说明,包括实验性的“做和不做”,并说明使用照片和电影的关键步骤,以帮助新研究人员建立这个有用的程序。
这个协议的一些优点和应用是:
1)时间控制可以通过分离期望阶段的胚胎海马来获得早期,中期或晚期妊娠祖细胞。
2)空间控制可以通过将电极定位为靶向海马体或皮质的不同区域来实现。
3)Cre-介导的重组可以通过将Cre-GFP电穿孔到携带期望的floxed等位基因的胚胎中来使用。
4)可以电穿孔过表达构建体。
5)胚胎致死菌株可以通过在生存窗口中进行操作来获得,然后可以在器官外植体中进一步发育。

关键字:小鼠海马, 胚胎, 电穿孔, 海马切片, 器官型外植体

材料和试剂

  1. 塑料巴斯德移液器:
    3毫升(P-3)和2毫升(P-1)(金牛座生物医学)
    1.5毫升移液器(RPI,目录号:147500)
    注:从今以后,这些都将被称为'移液器'。获得正确尺寸的移液管并将轴切割至正确的孔径尺寸(见图1)是操作大脑,半球和外植体而不损坏它们的关键。


    图1.塑料巴斯德移液器。 :一种。在此过程中使用不同大小的移液管:#1(1.5毫升移液管),#2(2毫升移液管),#3(3毫升移液管),如图所示切割#4。切割仅在移液管轴直径开始渐缩的位置进行,距离尖端约2 cm。 B.用移液器在酒精中消毒的烧杯。

  2. 35毫米无菌培养皿(Laxbro,目录号:PD-35 TC)
  3. 细胞培养6孔组织培养板(Thermo Fisher Scientific,目录号:140675)
  4. 微管尖端
  5. 玻璃毛细管:不含细丝的薄壁硼硅管(Sutter Instruments,目录号:B100-75-10)
  6. 100毫米培养皿(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:150464)
  7. 无菌刀片
  8. 细胞培养插入物:0.4μm(Millicell,Merck,目录号:PICM03050)
  9. 吸气器组件(Sigma-Aldrich,目录号:A5177-5EA)
  10. 带有0.22μm过滤器(Millex-GP,Merck,目录号:SLGP033RS)的50 ml注射器过滤器单元用于对培养基进行灭菌
  11. 2喷瓶(Tarsons,目录号:561100)(含70%和100%乙醇)
  12. pCS2-EGFP质粒(或任何所需的质粒)
  13. 怀孕的瑞士小鼠(SWR / J)(从塔塔基础研究所繁殖设施获得)
  14. 无水乙醇(Merck,目录号:107017)
  15. Leibovitz的L-15培养基(Thermo Fisher Scientific,Gibco TM,目录号:41300039)
  16. 快速绿色染料(西格玛奥德里奇,目录号:F7252)
  17. 青霉素 - 链霉素(P / S)(10,000U / ml)(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  18. Dulbecco改良的Eagle培养基(Thermo Fisher Scientific,Gibco TM,目录号:12800017)
  19. B27 TM补充物(50x),无血清(Thermo Fisher Scientific,Gibco TM,目录号:17504044)
  20. DMEM培养基的工作溶液(请参阅食谱)

设备

  1. 水平流罩(Kirloskar,Envair Electrodyne,目录号:KCH-B)
  2. 玻璃烧杯(Borosil,产品目录号:1000D21)
  3. 组织切碎机(McIlwain,型号:MTC / 2E)
  4. 立体显微镜(奥林巴斯,目录号:SZ61)置于水平流动组织培养罩
  5. 细胞培养孵育器(Thermo Fisher Scientific,型号:Heracell TM 150)
  6. 操纵胚胎大脑的精细工具:
    1. Dumont#5镊子,Dumostar(Roboz手术器械,目录号:RS-4978)
    2. Dumont#55,镊子,Dumostar(Roboz手术器械,目录号:RS-4984)
    3. 微解剖弹簧剪刀(Roboz手术器械,目录号:RS-5603)
  7. 用于处理斩波器光盘的粗镊子(Roboz手术器械,产品目录号:RS-5040)

  8. 用于打开大坝和去除子宫的精细剪刀(Fine Science Tools,目录号:14060-11)和组织钳(Roboz Surgical Instrument,目录号:RS-8166)
  9. 电极(3毫米,BTX,哈佛设备,目录号:450487)
  10. Electroporator(BTX,Harvard Apparatus,型号:ECM 830)

  11. 双级玻璃微管拉拔器(NARISHIGE,型号:PC-10)

程序

道德声明:所有程序都遵循Tata基础研究机构动物伦理委员会指导原则。

  1. 设置(预备)
    注意:收获胚胎后的所有步骤均在水平流罩中进行。
    1. 用70%乙醇擦拭组织培养罩的表面。
    2. 将70%的乙醇倒入200毫升的玻璃烧杯中,通过将乙醇一直吸入灯泡然后将其排出来对#1,#2和#3移液器进行消毒。让剩余的乙醇排出。
    3. 用L-15培养基清洗移液管以去除痕量的乙醇,并保持站立在解剖显微镜附近的无菌空烧杯中,以便于接近。
    4. 将解剖器械置于含有70%乙醇的塑料托盘中10分钟,然后在使用前擦干。
      在无菌托盘中放置,以便在操作过程中轻松进入
    5. 将大约六个35毫米的培养皿放在冰上填充L-15。
    6. 在需要数量的6孔组织培养板的孔中加入1.25ml的DMEM培养基。使用无菌镊子每孔放置一个无菌细胞培养插入物。将6孔板转移至37°C培养箱以预热培养基。通常,取决于胚胎阶段,5-8个器官型外植体可以容纳在一个细胞培养插入物上。

  2. 准备注射
    1. 在无核酸酶的水中准备2μg/μl的pCS2-EGFP质粒(或任何所需的质粒)。用微量吸头轻轻触摸一些快速的绿色染料,并将其引入DNA溶液中并混合。这样可以在注射期间和注射后观察DNA溶液。
    2. 为了准备玻璃毛细管,将毛细管拉拔器设置在62.5°C的温度下进行一步拉伸。
    3. 使用拉拔器拉动毛细管,并将它们放置在毛细管支架上的培养皿中。
    4. 将毛细管安装在吸嘴上吸取橡皮吸管,并吸取约5-10μl含有快速绿色染料的DNA溶液。这将持续大约2-3次体内注射。

  3. 收获胚胎
    1. 使用贵研究所许可的程序牺牲期望阶段的孕坝(E13.5-18.5)。下面显示的示例是瑞士鼠标的E15.5。在C57BL / 6J背景下的Nfia突变体胚胎也已成功用于2011年的Subramanian等人。,
    2. 用乙醇擦拭腹部皮肤,并做一个纵向切口。用70%乙醇(喷瓶)清洁切口区域,并使用一副新的无菌镊子和剪刀在腹壁上切开一个切口。
    3. 注意不要接触大坝的皮肤或头发,一个接一个地提起子宫的每个手臂。在最低点切下子宫并转移到含有无菌PBS的培养皿中。从这一点开始,所有程序都将在组织培养罩内进行。
    4. 使用解剖显微镜仔细地从子宫中取出胚胎,并将它们放入含有无菌1x PBS的无菌100mm培养皿中。

    5. 斩首胚胎并将头转移到含有L-15的新鲜无菌培养皿中。
    6. 解剖大脑并使用移液管#3将其转移至含有保存在冰上的L-15的35mm培养皿。去除大脑后,切断后脑和中脑,只保留包括嗅球在内的前脑。嗅球的存在有助于将远端半球以正确的方向放置在组织切碎器板上。只有在电击后才能保留和移除脑膜。
    注:多次转移(步骤C4-C6)到含有无菌PBS / L-15的新鲜培养皿中,有助于清除任何可能的污染并保持无菌状态。

  4. 电穿孔和器官切片制备
    图2显示了总体程序的总结漫画,以及培养期结束时的样本器官外植体。


    图2.体外电穿孔和器官切片制备的总结示意图A.注射DNA和完整脑电穿孔; B.含有电穿孔祖细胞的切片(绿色圆圈); C.在体外6天后,外植体显示存在于锥体细胞层(黄色)内的表达GFP的锥体神经元(绿色三角形)。这些神经元最初延伸轴突(绿色),朝向发育中的毛细胞(箭头)生长,然后环绕外植体的外围,可能是因为它们不能将毛细管离开过滤器。 D.一种器官外植体的图像,显示轴突束生长到菌毛中,然后在外植体周围(箭头)。比例尺=100μm。该图和图例从图5 Subramanian et al。,PNAS,2011年进行了修改。

    1. 将组织切碎机设置为250μm,并将无菌刀片插入刀片。拧紧刀片。
      使用喷射瓶将100%乙醇冲洗在刀片上并让设备干燥。
    2. 用乙醇擦拭桨叶电极并将它们连接到electroporator。
    3. 将参数设置为以下内容:电压50 V;时间50毫秒;脉冲数5;脉冲间隔999毫秒。
    4. 如图3A所示,将半球的心室注入所需的DNA溶液(与Fast Green混合)电穿孔。心室是中空的,可以通过其较暗的外观来识别,并且当微量吸管刺穿神经上皮并进入心室时阻力减小。建议进行单侧注射和电穿孔,然而,染料-DNA溶液有时可能扩散到连接的对侧脑室(见图3B)。这是正常的,不影响实验设置。用溶液填充侧脑室,直到染料显示心室腔被填充(见图3B)。取决于使用的胚胎的年龄,可以将大约5-10μl的溶液注入心室。由于DNA注入心室,最容易进入DNA的细胞是心室区细胞,其由分裂的神经元祖细胞组成。这是电穿孔的主要人群(Saito,2006)。


      图3.注射DNA溶液到远端脑半球和电穿孔。A.玻璃毛细管(箭头)将DNA注入心室腔。 B.桨叶电极(正极和负极)以适合于电穿孔海马的角度定位。 ( *在图3B中,负电极的绝缘物在尖端被削去,所以一部分桨叶呈现银色,这不影响电穿孔。)比例尺=500μm。 />
    5. 立即进行电击,使DNA溶液不会扩散到心室外。将电极放置在图3B所示的角度,使正极位于对侧,负极位于半球的同侧以进行电穿孔。由于DNA带负电荷,它会向正电极移动,因此在半球的内侧显示最大的电穿孔。
    6. 启动electroporator上的脉冲序列。电穿孔区域周围的桨叶下面的气泡指示电流成功通过。 ( - )桨式电极下通常会出现更多气泡。
    7. 将电穿孔的大脑转移到含有冰冷的L-15培养基的35mm培养皿中。保持冰5分钟。与此同时,根据实验要求电穿孔其他大脑。将每只大脑转移到冰冷的L-15上。
    8. 一旦所有需要的大脑都被电穿孔,分离电穿孔的半球并仔细去除脑膜。
    9. 将半球放置在组织切碎器上,使得内侧面与台上的白色圆盘接触,并且侧面(新皮层)面朝上。使用移液器#1轻轻地移除多余的培养基。过量的介质会导致半球在下一步(切碎)中摆动。在吸取多余的培养基的同时,使半球的尾轴与叶片正交,以便获得冠状切片(嗅球需要指向右或左,而不是朝向或远离你,如图4所示)。


      图4.将解剖的电穿孔半球放置在组织切碎器的台上。 请注意,将砧板光盘固定在位的弹簧夹应朝向R-L方向,以防止它们与刀片发生干涉。

      头脑半球的方向应该被考虑在内(嗅球指向R或L)。

    10. 开始斩波(视频1和2)。立即用两对强力钳取出夹持切碎组织的圆盘。将组织放入含有冷L-15(视频3)的清洁含L-15的碟中。

      s视频1

      s视频2

      s视频3

    11. 半球看起来像一块切片面包(图5A)。翻转半球,使内侧面朝上。轻轻地刺激以从内侧释放指状突起,其是海马的单个切片。使用精细的镊子从新皮层切断海马(虚线,图5A)。在含有冰冷的L-15培养基的新鲜35毫米培养皿中收集海马切片。


      图5.从切碎的半球中分离器官型海马切片。 :一种。显示“切片面包”外观的切碎半球的图像。 B.从切碎的半球分离出单个海马外植体。 C.(B)中的相同外植体重新定向并标记。 CA1,CA3,海马田地。 DG,齿状回。比例尺= 500微米。

    12. 从培养箱中取出含有细胞培养插入物的6孔培养皿并置于解剖显微镜下(图6)。


      图6.具有细胞培养插入物的6孔组织培养皿

    13. 使用移液管#2吸取5-6个海马外植体与一些L-15培养基。通过轻轻地向移液管的球管施加压力,将外植体逐个放到滤盘上,以便只分配一个外植体,每个都在其自身的L-15培养基液滴中(图7A)。使用移液管#1轻轻吸出多余的培养基,并使用吸液产生的流体流动轻轻定位外植体,使它们互不接触,也不会粘在细胞培养插入物的壁上(图7B)。应该有足够的培养基来防止外植体变干,但应避免使用过量的培养基,以防止外植体折叠或漂浮(图7C和7D)。外植体不会干燥;他们可以通过过滤器访问井中的DMEM培养基。


      图7.将海马外植体置于培养插件上。 :一种。使用移液管#2将外植体置于单独的培养基液滴上。 B.使用移液器#1移除多余的液体。在这个步骤中,可以分离两个可能已经沉积在相同液滴中的外植体(C)。 D.程序完成时外植体的图像。

    14. 在37℃,5%CO 2下孵育海马外植体。每隔一天更换媒体。
    15. 几个小时内,如果使用荧光报告基因构建物,电穿孔可以被显现。在接下来的3-5天内,标记的祖细胞分化成锥体神经元,将顶端过程延伸至卷曲外植体的中心,并且轴突沿着外植体到达伞形区域(图8)。


      图8.在连续的天内在体外成像的单个器官型海马体外植体 A.在体外1天后(DIV),电穿孔的细胞是看到靠近心室区(黄色虚线)。顶端过程看起来延伸到外植体箭头的中心)。 (B)和(C)在体外进行额外的天时,细胞开始从体内迁移离开心室区(黄色箭头)并形成金字塔形细胞层。 D.通过体外6天,电穿孔的细胞延伸轴突(白色箭头),可以看到围绕外植体。 (A-D)中的图像是由多个拼接在一起的帧组成的复合物。比例尺= 100微米。

数据分析

这种体外电穿孔的海马器官制剂可用于评估神经元或神经胶质是否由祖细胞产生。在伞毛中存在表达GFP的轴突是神经发生的指示。参见Subramanian等人(2011),图5,用神经原或神经胶质细胞构建体电穿孔的外植体图像。这些外植体也可以针对细胞类型特异性标记进行免疫染色并根据需要分析兴奋性/抑制性神经元或神经胶质的分布。

笔记

  1. 重现性和可变性:电穿孔的程度是可变的,但可以通过练习使其更加可重复。需要注意的关键问题是电极的角度以及人们是如何小心地控制大脑即组织与桨叶电极的接触。
    在电穿孔过程中,大脑需要完全浸没在培养基中。
  2. 技术提示和注意事项:
    1. 生产外植体的速度对外植体健康至关重要。
      。当组织不在显微镜或切碎阶段时,将组织保存在冰冷的L-15上很关键。
    2. 能够在切碎的大脑中识别“良好”的海马状指状突起也伴随着练习。通常,在切碎的脑的尾端可以看到易于识别的海马形态,而从更多内侧位置制备的外植体只能通过微小的菌毛识别。一个很好的经验法则是,如果它没有一个伞,它可能不是一个海马外植体,而是一个弯曲的新皮层伪装成一个海马。
    3. 细胞培养插入物最适合于6孔板。不要尝试使用单个35毫米的碟子 - 它们没有必要的高度。这道菜不能正常关闭,所以介质会干燥或被污染。

食谱

  1. DMEM培养基的工作溶液(50毫升)
    B27:500微升
    青霉素 - 链霉素:500μl的100x股票
    DMEM:49毫升
    使用0.22μm过滤器过滤灭菌,并在4°C下保存。按照B27
    文档手册中的建议,使用此解决方案长达1周

致谢

我们感谢Leora D'Souza协助录像,S。Suryavanshi博士和TIFR动物繁殖设施提供出色的支持。该协议是在Tole实验室利用来自Wellcome Trust高级研究员(056684 / Z / 99 / Z),Swarnajayanti研究员(4/3/2005-SF)的支持,印度政府生物技术部的赠款和从TIFR-DAE到ST的校内资金;来自TIFR捐赠基金的Kanwal Rekhi职业发展奖给LS。 ST是Shanti Swarup Bhatnagar奖(印度政府科学与工业研究委员会)和Infosys生命科学奖(Infosys科学基金会)的获得者。
该方案用于Subramanian等人(2011)Proc Natl Acad Sci U S A 108(27):E265-274。 (现在的地址:Eli& Edythe Broad Center of Regeneration Medicine& Stem Cell Research,University of California San Francisco,CA 94143,USA)。
作者没有利益冲突或利益冲突。

参考

  1. Opitz-Araya,X.和Barria,A。(2011)。 器官型海马切片培养。 Exp Exp (48 )。
  2. Saito,T.(2006)。 体内电穿孔胚胎鼠中枢神经系统 Nature Protoc 1(3):1552-1558。
  3. Stoppini,L.,Buchs,P.A。和Muller,D。(1991)。 一种用于神经组织器官培养的简单方法 Neurosci Methods < 37(2):173-182。
  4. Subramanian,L.,Sarkar,A.,Shetty,AS,Muralidharan,B.,Padmanabhan,H.,Piper,M.,Monuki,ES,Bach,I.,Gronostajski,RM,Richards,LJ和Tole,S. (2011年)。 转录因子Lhx2对于抑制星形胶质细胞生成和促进发育中海马的神经发生是必要和充分的。美国国立科学院美国科学院 108(27):E265-274。
  5. Tole,S.,Christian,C.和Grove,E.A。(1997)。 早期规范和自主开发小鼠海马皮质区域 开发 124(24):4959-4970。
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引用:Iyer, A., Subramanian, L. and Tole, S. (2018). Organotypic Explants of the Embryonic Rodent Hippocampus: An Accessible System for Transgenesis. Bio-protocol 8(6): e2764. DOI: 10.21769/BioProtoc.2764.
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