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Neurosciences

宫内电穿孔诱导小鼠大脑皮质中氧合酶的重组

Published: November 17, 2017
doi:
10.3791/56675
, , , ,
1Department of Biology,James Madison University

Summary

在稀疏的细胞群中, 可以通过诱导功能的丧失或增益来研究脑内基因的细胞自主功能。在这里, 我们描述在子宫内电穿孔, 以提供重组到稀疏的人群发育皮层神经元与 floxed 基因, 导致功能丧失在体内。

Abstract

基因的细胞自主神经元功能可以通过在小的和稀疏的神经元中造成基因功能的丧失或增益而显露出来。要做到这一点, 就需要生成一个镶嵌体, 其中基因的功能丧失或增益的神经元被不受基因扰动的组织包围。在这里, 我们结合了综合考试-液氧复合系统与在宫内电穿孔, 以产生马赛克脑组织, 可用于研究细胞自主功能的基因在神经元。DNA 结构 (可通过存储库提供), 编码的荧光标签和重组, 被引入到开发的皮层神经元含有基因的 loxP 部位的大脑中的小鼠胚胎使用在子宫穿孔.此外, 我们描述了各种适应的子宫内电穿孔方法, 提高生存能力和再现性。该方法还包括建立一个在稀疏或稠密的神经元中的重组的效价。标记脑组织的组织学准备不需要 (但可以适应) 免疫组化。该结构的使用保证了荧光标记神经元携带的基因重组。组织学准备可以通过对树突和轴突和树突棘的共聚焦成像对神经元进行形态学分析。由于稀疏镶嵌组织中功能的丧失或增益达到了目的, 因此, 该方法允许研究基因产品的细胞自主性和足够度在体内。

Introduction

生成一个遗传马赛克是一个经典的实验范式, 以了解的功能基因的兴趣。为了确定基因是否是细胞表型的必要条件, 最简单的方法是在整个生物体中导致基因功能的丧失 (例如击倒)。然而, 要确定某一基因是否需要特定的细胞类型, 基因在整个生物体中的击倒并不是一种有效的方法。相反, 一个方法是需要的, 这将导致在一个给定的细胞中的基因功能丧失, 而它是由野生 (即不受基因干扰) tissue-in 换句话说, 创造马赛克组织。如果突变细胞显示出突变的表型, 但周围的野生细胞没有, 该基因功能的细胞自主的方式。分析的马赛克组织, 其中突变细胞周围的野生组织, 是理想的理解细胞自主功能的基因, 特别是在大脑中的神经元和神经胶质形成一个巨大的互联网络的组织。

多种形式的马赛克脑组织为研究基因的细胞自主功能提供了强有力的模型。研究的重点是神经元移植1, 女性 X 链接嵌2,3,4, 以及内生体细胞嵌5,6根据马赛克得出了他们的结论脑组织。有条件地删除基因通过 “氧合酶复合系统” 是一种充分利用转基因小鼠品系的方法。在这个方法中, 两个 loxP 站点被引入到一个必需的基因序列的两侧 (如外显子), 它的两侧是 loxP 的站点, 它们都朝同一方向 (“floxed”)。重组将 loxP 站点之间的顺序7。通过将 floxed 小鼠转到另一条表达重组的老鼠线上, 并在细胞子集 (“记者行”) 中使用荧光标记, 可以实现对重组的再组合。这已被证明在各种方法, 以揭示的功能, 一个基因的细胞子集, 如兴奋性神经元或胶质细胞8。记者线可以表达 CreER 的T2 , 以允许由药物诱导的重组 (神经元标记与诱导的综合治疗的淘汰赛, 或光滑)9。在另一种称为 “镶嵌分析双标记 (决策)”10,11的策略中, interchromosomal 重组允许与杂组织一起创建一个纯合突变体。在这些方法中, 每个候选基因或细胞亚型都需要在每次测试时产生一条新的小鼠。另外, 通过导入12或通过病毒载体 (例如腺相关病毒13或慢14携带特定于细胞的子类型, 可以引入后天重组。发起人)。这一策略产生强烈的产后标记。为靶向发育的脑皮质神经元稀少和产前, 一个理想的策略是宫内电穿孔重组与荧光标记。

除了通过在子宫内电穿孔, 通过在体内生成马赛克组织, 我们还对其他已发布的协议15、16中的过程进行了一些改编. 17,18,19,20,21。我们提供信息, 以提高成功繁殖定时怀孕的女性。我们还概述了在皮层组织中引入稀疏和明亮的神经元标记的两个策略: 一种策略是滴定重组的单个构造编码的级别和一个荧光标记22。另一种策略是使用 “超新星” 系统, 这些参数专门设计为23,24。此外, 我们提供了改进的生产一致的显微注射管和简化的在子宫内电穿孔手术。最后, 我们概述的关键步骤, 简化组织学准备, 允许分析树突棘和树突和轴突乔木, 没有进一步染色或免疫组化。

Protocol

这里描述的方法已经得到了詹姆斯麦迪逊大学动物护理和使用委员会 (ACUC) 的批准, 并符合所有相关的法规和机构准则。 1. 鼠标设置 房子一个年轻的 (> P60) 男性和女性合 floxed 小鼠一起建立一个饲养员对25。注意: 一个好的负控制是建立一个额外的增殖对野生小鼠, 其中综合重组表达不会导致马赛克26。 让雌鸟生下她的头, ?…

Representative Results

单一结构的 GFP。electroporated 在 E15.5 和可视化的 P14。根据结构的浓度和注入量, 可以获得稀疏或稠密的结果22,26。例如, 注射1µL 2 毫克/毫升 GFP。考试结果在稀疏分布的标记细胞, 其中一些可以是明亮的 (图 1A), 并本地化第二层/III (图 1B)。由于组织是100µm 厚, 大多数树突乔木保存 (<strong…

Discussion

在这里, 我们介绍了在子宫内电穿孔与重组在 floxed 小鼠的组合, 以产生马赛克脑组织。这种方法的优点是, 每次要针对不同的蜂窝子类型时都不需要生成新的鼠标线:在子宫内电穿孔可用于针对兴奋性神经元、抑制性神经元或胶质细胞, 这取决于时间以及电穿孔的位置15,16,17,18,<sup c…

Divulgations

The authors have nothing to disclose.

Acknowledgements

作者感谢詹姆斯麦迪逊大学生物学系和詹姆斯麦迪逊大学光显微镜和成像设施的慷慨支持。Dr. 马克 l. 加布里埃尔关于年轻的产后组织准备的有用建议,和 Drs. 贾斯汀 w. 布朗和科里. 克莱兰德为外科手术材料和空间的慷慨协调。这项研究部分由 4 VA 合作研究基金资助, 这是一个促进弗吉尼亚联邦 (G.S.V.) 的合作伙伴关系, 以及弗吉尼亚科学院小项目研究补助金 (G.S.V.)。支持已慷慨提供的贝蒂祖爱管家58的本科研究奖学金 (K.M.B.), 法雷尔暑期研究奖学金 (K.M.B.), 詹姆斯麦迪逊大学第二世纪奖学金 (K.M.B.), 詹姆斯麦迪逊大学百年奖学金 (到 C.J.H.), 詹姆斯麦迪逊大学露西鲁宾逊查寻30纪念奖学金 (对 Z.L.H.) 和詹姆斯麦迪逊大学科学和数学学院协助津贴 (对 G.S.V.)。

Materials

C57BL/6J mice The Jackson Laboratory #000664 See "1. Mouse set-up" (step 1.1, "wildtype mice")
GFP.Cre empty vector AddGene #20781 See "2. DNA set-up" (step 2.1 "single DNA construct that codes for Cre recombinase as well as a fluorescent marker"). GFP.Cre empty vector was a gift from Tyler Jacks.
pK029.CAG-loxP-stop-loxP-RFP-ires-tTA-WPRE (Supernova) AddGene #69138 See "2. DNA set-up" (step 2.1 "Supernova" system) and http://snsupport.webcrow.jp/. pK029.CAG-loxP-stop-loxP-RFP-ires-tTA-WPRE (Supernova) was a gift from Takuji Iwasato.
pK031.TRE-Cre (Supernova) AddGene #69136 See "2. DNA set-up" (step 2.1 "Supernova" system) and http://snsupport.webcrow.jp/. pK031.TRE-Cre (Supernova) was a gift from Takuji Iwasato.
pK038.CAG-loxP-stop-loxP-EGFP-ires-tTA-WPRE (Supernova) AddGene #85006 See "2. DNA set-up" (step 2.1 "Supernova" system) and http://snsupport.webcrow.jp/. pK038.CAG-loxP-stop-loxP-EGFP-ires-tTA-WPRE (Supernova) was a gift from Takuji Iwasato.
EndoFree Plasmid Maxi Kit (10) Qiagen #12362 See "2. DNA set-up" (step 2.3 "endotoxin-free plasmid purification kit")
Trypan Blue powder, BioReagent grade Sigma T6146-5G See "2. DNA set-up" (step 2.5 "trypan blue")
Sodium Chloride, ACS, 2.5 kg VWR BDH9286-2.5KG See "2. DNA set-up" (step 2.5 "NaCl")
Potassium Chloride, ACS, 500 g VWR #97061-566 See "2. DNA set-up" (step 2.5 "KCl")
Sodium phosphate dibasic, ReagentPlus, 100 g Sigma-Aldrich S0876-100G See "2. DNA set-up" (step 2.5 "Na2HPO4")
Potassium phosphate monobasic, ReagentPlus, 100 g Sigma-Aldrich P5379-100G See "2. DNA set-up" (step 2.5 "KH2PO4")
Hydrochloric acid, ACS reagent, 500 mL Fisher Scientific A144-500 See "2. DNA set-up" (step 2.5 "HCl")
P-97 Micropipette Puller Sutter Instrument P-97 See "3. Pipette set-up" (step 3.1 "glass capillary puller")
3.0 mm wide trough filament Sutter Instrument FT330B See "3. Pipette set-up" (step 3.1 "glass capillary puller")
Thin Wall Glass Capillaries, 4", 1 / 0.75 OD/ID World Precision Instruments TW100-4 See "3. Pipette set-up" (step 3.1.1 "glass capillary")
Single Ply Soft-Tech Wipes, 4.5" Phenix LW-8148 See "3. Pipette set-up" (step 3.2.1 "single-ply task wipe"); other single-ply wipes (e.g. Kimwipes) can be used.
Graefe Forceps, 7 cm, Straight, 0.7 mm 1×2 Teeth World Precision Instruments #14140 See "4. In utero electroporation" (step 4.1 "Graefe forceps")
Iris Scissors, 11.5 cm, Straight, 12-pack World Precision Instruments #503708-12 See "4. In utero electroporation" (step 4.1 "iris scissors")
Hartman Mosquito Forceps, 9 cm, Straight, 12-pack World Precision Instruments #503728-12 See "4. In utero electroporation" (step 4.1 "Hartman mosquito forceps")
General Purpose Non-Woven Sponges, 2" x 2", 4-ply Medrepexpress #2204-c See "4. In utero electroporation" (step 4.1 "non-woven gauze sponges")
Ring Tipped Forceps, 10 cm, Straight, 2.2mm ID World Precision Instruments #503203 See "4. In utero electroporation" (step 4.1 "ring-tipped forceps")
Pyrex petri dishes complete, O.D. × H 100 mm × 20 mm Sigma-Aldrich CLS3160102-12EA See "4. In utero electroporation" (step 4.1 "Petri dishes")
Flat Type Instrument Tray, Stainless Steel, 13-5/8" x 9-3/4" x 5/8" Amazon B007SHGAHA See "4. In utero electroporation" (step 4.1 "stainless steel tray")
Platinum Tweezertrode, 5 mm BTX #45-0489 See "4. In utero electroporation" (step 4.3 and 4.16 "tweezer-type electrodes")
ECM 830 Foot Pedal BTX #45-0211 See "4. In utero electroporation" (step 4.3 and 4.17 "foot pedal")
ECM 830 Generator BTX #45-0052 See "4. In utero electroporation" (step 4.3 "generator")
Single Animal Isoflurane Anesthesia System with Small Induction Box Harvard Apparatus #72-6468 See "4. In utero electroporation" (step 4.4 and 4.6 "nose cone", step 4.4 "induction chamber")
Ophthalmic ointment Hanna Pharmaceutical Supply Co #0536108691 See "4. In utero electroporation" (step 4.7 "veterinary ophthalmic ointment")
Space Gel (AIMS) VWR #95059-640 See "4. In utero electroporation" (step 4.8 "sealed pouch filled with supersaturated salt solution")
Hair Remover Gel Cream, Sensitive Formula Veet #062200809951 See "4. In utero electroporation" (step 4.9 "depilatory cream")
10ul Low Retention Tip Starter (960 tips/pk) Phenix Research Products TSP-10LKIT See "4. In utero electroporation" (step 4.12 "sterile 10 µL micropipette tip")
Aspirator tube assemblies for calibrated microcapillary pipettes Sigma-Aldrich A5177 See "4. In utero electroporation" (step 4.15 "aspirator tube assembly")
Braided Absorbable Suture, 4-0, Needle NFS-2(FS-2), 27" Medrepexpress MV-J397 See "4. In utero electroporation" (step 4.19 "absorbable sutures")
“LiquiVet Rapid” Tissue Adhesive Medrepexpress VG3 See "4. In utero electroporation" (step 4.20 "tissue adhesive")
Hypodermic syringes, polypropylene, Luer lock tip, capacity 1.0 mL Sigma-Aldrich Z551546-100EA See "4. In utero electroporation" (step 4.21 "1 mL syringe")
BD Precisionglide syringe needles gauge 26, L 1/2 in. Sigma-Aldrich Z192392-100EA See "4. In utero electroporation" (step 4.21 "26G, ½” needle")
Nestlets Nesting Material Ancare NES3600 See "4. In utero electroporation" (step 4.24 "nesting materials")
Sunflower Seeds, Black Oil, Sterile Bio-Serv S5137 See "4. In utero electroporation" (step 4.24 "sunflower seeds")
Paraformaldehyde, 97% Alfa Aesar A11313 See "5. Histology" (step 5.1.1 "PFA")
Economy Tweezers #3, 11 cm, 0.2 x 0.4 mm tips World Precision Instruments #501976 See "5. Histology" (step 5.5 "tweezers")
Agar powder Alfa Aesar #10752 See "5. Histology" (step 5.8.1 "agar")
Single-edge razor blades, #9 blade Stanley Tools #11-515 See "5. Histology" (step 5.9 "single-edge razor blade")
Specimen disc S D 50 mm Leica #14046327404 See "5. Histology" (step 5.9 "vibrating microtome specimen disc")
Buffer tray S assembly Leica #1404630132 See "5. Histology" (step 5.10 "buffer tray")
VT1000 S Vibratome Leica #14047235612 See "5. Histology" (step 5.10 "vibrating microtome")
Double Edge Razor Blades Personna BP9020 See "5. Histology" (step 5.10 "blade")
Knife Holder S Leica #14046230131 See "5. Histology" (step 5.10 "knife holder")
Studio Elements Golden Taklon Short Handle Round Brush Set Amazon B0089KU6XE See "5. Histology" (step 5.12.1 "fine tipped paintbrush")
Superfrost Plus Slides Electron Microscopy Services #71869-11 See "5. Histology" (step 5.12.1 "microscope slide")
ProLong Diamond Antifade Mountant, 10 ml Thermofisher P36970 See "5. Histology" (step 5.12.3-5.12.4 "mountant")
Cover Glass, 24 x 50 mm, No. 1 Phenix Research Products MS1415-10 See "5. Histology" (step 5.12.4 "coverslip")
4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) Sigma-Aldrich D9542 See "5. Histology" (step 5.13.1 "DAPI")
Fixed Stage Upright Microscope Olympus BX51WI See "5. Histology" (step 5.15 "light microscope")
Laser Scanning Confocal Microscope Nikon TE2000/C2si See "5. Histology" (step 5.15 "confocal microscope")
4x objective, NA = 0.20 Nikon CFI Plan Apo Lambda 4X See "5. Histology" (step 5.15 "low-power objective")
20x objective, NA = 0.75 Nikon CFI Plan Apo Lambda 20X See "5. Histology" (step 5.15 "medium-power objective")
60x objective, NA = 1.40 Nikon CFI Plan Apo VC 60X Oil See "5. Histology" (step 5.15 "high-power objective")
DOWNLOAD MATERIALS LIST

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Tags

Cre-lox RecombinationMouse Cerebral CortexIn Utero ElectroporationGenetic MosaicNeurobiologyLayer 2-3 Cortical NeuronsPipette PreparationEmbryonic Day 15.5AnesthesiaMouse Surgery

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Bland, K. M., Casey, Z. O., Handwerk, C. J., Holley, Z. L., Vidal, G. S. Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation. J. Vis. Exp. (129), e56675, doi:10.3791/56675 (2017).
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Réimpressions et Autorisations

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