小鼠卵母细胞在减数分裂过程中对人类基因功能的评估
Summary
随着与人类疾病相关的遗传变异开始显露出来, 开发系统以迅速评估那些被确定的变种的生物学意义变得越来越重要。本协议描述了在女性减数分裂过程中使用小鼠卵母细胞评估人类基因功能的方法。
Abstract
胚胎体检是人类不孕症的主要遗传原因。这些事件大多来源于女性减数分裂, 尽管与孕产妇年龄呈正相关, 但年龄并不总是预示着产生异倍体胚胎的风险。因此, 基因变体可能会解释在卵子发生过程期间染色体分离的错误。鉴于对人类卵母细胞的访问有限的研究目的, 通过一系列的分析, 以研究人类基因功能在减数分裂 I 使用小鼠卵母细胞。首先, 基因的信使 RNA (mRNA) 和感兴趣的基因变异被杏仁入前期 I 被拘捕的老鼠卵母细胞。在允许表达时间后, 卵母细胞被同步释放到减数分裂成熟完成减数分裂 i。通过用荧光记者的序列标记 mRNA, 如绿色荧光蛋白 (Gfp), 除了表型改变外, 还可以评估人体蛋白的定位。例如, 可以通过建立挑战基因产物来修复减数分裂错误的实验条件来调查功能的增益或丧失。虽然这一系统有利于在卵子发生过程期间对人体蛋白质功能进行调查, 但由于蛋白质表达不在内源性水平, 而且除非受控(即敲), 小鼠同系物也存在于系统中。
Introduction
不孕是影响10-15% 人口的生育年龄1的一个条件, 近半数的人寻求医疗治疗2。虽然不孕的病因是多种多样的, 在许多情况下多因素, 最常见的遗传异常的人是胚胎体检3。体检被定义为细胞中正确数目的染色体的偏差 (增益或损耗)。体检在人类胚胎中的现象是常见的, 并随着高级孕产妇年龄的增长而增加4,5。四项随机对照试验突出了仅选择染色体正常 (euploid) 胚胎进行子宫移植的好处, 因为这一策略导致植入率增加, 流产率降低, 并且缩短了实现怀孕6,7,8,9。因此, 了解人类体检的病因学对辅助生殖有重要意义。
虽然植入前的 aneuploidies 基因检测对不孕症治疗是有益的, 但对 aneuploidies 起源的深入了解仍然缺乏。人们普遍认为, 减数分裂 aneuploidies (起源于配子生产期间) 与孕产妇年龄呈正相关, 然而, 有些妇女呈现出与其特定年龄的平均率相差4的胚胎体检率。这些病例表明, 单独年龄并不总是预示着产生异倍体胚胎的风险。其他因素可能会在增加胚胎体检的风险方面发挥作用, 如基因变异。
在卵母细胞减数分裂过程中, 研究基因变异对体检的潜在贡献的一个关键方面是设计一个快速评价减数分裂基因功能的系统。由于道德约束和有限的访问, 使用人类卵子进行这些实验是不切实际的。这些问题可以通过使用小鼠卵母细胞来规避, 这里描述了在减数分裂过程中评估人类基因功能的一系列检测方法。通过 microinjecting 的信使 RNA (mRNA) 编码的基因变异, 在小鼠卵中的人蛋白的定位可以可视化, 并用于确定野生型和变异人蛋白的异位表达是否导致任何可能导致体检的表型改变。这些表型包括增加微管, 附加到不适当的修女动粒和无法支持染色体排列在中期的减数分裂 i. 重要的是, 这个协议可以用来调查的增益和损失的功能遗传变异通过建立特定的实验条件来挑战卵母细胞减数分裂的关键事件, 如纺锤体构建和染色体对齐10。
Protocol
1. 分子克隆 获得感兴趣基因的全长编码序列和质粒体外转录 (pIVT) 向量11。注: 全长 cDNA 克隆可从各种供应商处获得, 也可通过反向转录聚合酶链反应 (RT PCR) 产生。国家生物技术信息中心 (NCBI) 在线资源提供核苷酸和表达序列标记 (EST) 数据库的基因转录序列。为了便于蛋白质可视化和表达水平分析, 克隆策略中可能需要融合到绿色荧光蛋白 (Gfp) 或其他合适?…
Representative Results
在体外转录高品质的 RNA 将有一个 A260/A280 比 (1.8-2.2) 和 A260/A230 比≥1.7 时使用分光光度计。图 1中的图片显示了在电泳后变性琼脂糖凝胶上体外产生的 RNA 的迁移。在图案中涂抹的带或具有多个尺寸波段的样本可以指示样品的污染或降解。另外, 多波段可能表明 mRNA 不是完全变性, 或起始质粒没有完全线性化。mrna 的运行比预测的要大, 因?…
Discussion
由于与疾病有关的人类遗传变异的快速和不断增加的识别, 必须建立系统来评估其生物学意义。了解人类减数分裂中的蛋白质功能会带来特殊的挑战, 因为人类卵母细胞是珍贵的, 稀有的, 人类的精子不能服从基因的操纵。小鼠卵母细胞是哺乳动物模型系统, 对评估人类减数分裂基因功能有价值10,23。这个模型绕过了使用人类卵子的不切实际之处, 同时提供…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了来自美国生殖医学协会的研究补助金, 以及来自罗格斯大学的查尔斯和乔安娜. 布希纪念基金的支持, 新泽西州的 k.s.a. L.N. 得到了美国 (F31 HD0989597) 的资助。
Materials
| 0.2 mL Seal-Rite PCR tube | USA Scientific | 1602-4300 | |
| 1 kb DNA Ladder | Thermo Scientific | SM0313 | |
| 100 bp DNA Ladder | Thermo Scientific | SM0243 | |
| 6X DNA loading Dye | Thermo Scientific | R0611 | |
| 9-well glass spot plate | Thomas Scientific | 7812G17 | |
| Agarose | Sigma Aldrich | A9539 | |
| Albumin from bovine serum | Sigma-Aldrich | A3294 | |
| Alexa-fluor-568 conjugated anti-mouse IgG | Thermo Scientific | A21050 | 1:200 dilution |
| Alexa-fluor-633 conjugated anti-human IgG | Thermo Scientific | A21091 | 1:200 dilution |
| Ampicillin | VWR | AA0356 | |
| Anti-vibration table | Technical Manufacturing Corp | any standard model | |
| Anti-Acetylated Tubulin antibody | Sigma Aldrich | T7451 | 1:100 diution |
| Anti-centromeric (CREST) antibody | Antibodies Incorportated | 15-234 | 1:30 dilution |
| Barrier (Filter) Pipette tips | Thermo Scientific | AM12635 | Make sure compatable with your brand of pipettors. These are compatible with Eppendorf brand pipettors. |
| BD Difco Dehydrated Culture Media: LB Agar, Miller (Luria Bertani) | Fisher Scientific | DF0445-07-6 | |
| BD Difco Dehydrated Culture Media: LB Broth, Miller (Luria Bertani) | Fisher Scientific | DF0446-07-5 | |
| Capillary tubing | Sutter | B100-75-10 | |
| Center Well organ culture dish | VWR | 25381-141 | |
| CO2 tank | For incubator | ||
| Confocal microscope | Zeiss | any standard model | |
| Centrifuge (With cooling ability) | Thomas Scientific | any standard model | |
| Cover Glass 11 x 22 mm | Thomas Scientific | 6663F10 | |
| Coverslips | Thomas Scientific | 6663-F10 | thickness will vary for particular microscopes |
| DAPI | Sigma Aldrich | D9542 | |
| DEPC H20 | Life Technologies | AM9922 | |
| Digital Dry Bath | Thermo Scientific | 888700001 | |
| Easy A high fidelity cloning enzyme | Agilent | 600400 | For DNA cloning |
| Enzymes for linearizing pIVT | New England Biolabs | NdeI or KasI can be used | |
| Ethidium Bromide | Thermo Scientific | 155585011 | |
| Fluorescent Microscope | Any Fluorescent microscope may be used | ||
| Formaldehyde (37%) | Thermo Fisher Scientifc | 9311 | |
| Formaldehyde RNA loading dye | Ambion | 8552 | |
| Frosted Microscope Slides (Uncharged) 25X75 mm | Fisher Scientific | 12-544-3 | |
| Full Length cDNA Clones | Can be obtained from any vendor that supplies open reading frame clones | ||
| Gel electrophoresis apparatus | Bio-Rad | any standard model | |
| Glass Pasteur Pipets | Fisher Scientific | 13-678-200 | |
| Globin Forward Primer for pIVT Construct | 5'- GAA GCT CAG AAT AAA CGC -3'. Can be purchased from any company that generates custom oligonucelotides | ||
| Globin Reverse Primer for pIVT Construct | 5'- ATT CGG GTG TTC TTG AGG CTG G -3' Can be purchased from any company that generates custom oligonucelotides | ||
| Holding pipettes | Eppendorf | 930001015 | Vacutip |
| Humidified Chamber | Tupperware can be used | ||
| Illustra Ready-To-Go RT-PCR beads | GE Life Sciences | 27925901 | |
| Incubator | any standard model with CO2 and water jacketed technology | ||
| Inverted Microscope | Nikon instruments | Any Standard model | |
| Image J (NIH) Software | NIH | Image Analysis software | |
| Lid of 96 well plate | Nalgene Nunc International | 263339 | |
| Low Adhesion 0.5 mL microcentrifgue tube | USA Scientific | 1405-2600 | |
| MacVector | MacVector | Sequence analysis software | |
| MG132 | Selleckchem | S2619 | |
| Microscope slides | Fisher Scientific | 12-544-3 | |
| Millenium RNA Markers-Formaldehyde | Ambion | AM7151 | |
| Milrinone | Sigma-Aldrich | M4659 | Resuspend in DMSO at 2.5mM |
| Mineral Oil | Sigma-Aldrich | M5310 | Only used embryo-tested, sterile-filtered |
| Monastrol | Sigma-Aldrich | M8515 | Resuspend in DMSO at 100 mM |
| Mouthpiece | Biodiseno | MP-001-Y | |
| N2 tank | for antivibration table | ||
| Nail Polish; Clear | Any clear nailpolish can be used | ||
| NanoDrop Microvolume UV-Vis Spectrophotometer | Thermo Scientific | any standard model | |
| NorthernMax 10X Denaturing Gel Buffer | Life Technologies | AM8676 | |
| NorthernMax 10X Running buffer | Life Technologies | AM8671 | |
| NuPAGE MOPS SDS Running buffer | Thermo Scnentific | NP0001 | |
| Organ Culture Dish 60x15mm | Life Technologies | 08-772-12 | |
| Paraformaldehyde | Polysciences, Inc. | 577773 | |
| PCR Thermal Cycler | Thermo Fisher Scientific | 4484075 | |
| Petri Dish 139 mm | Thermo Fisher Scientifc | 501V | |
| Petri dish 35 mm | Thermo Fisher Scientifc | 121V | |
| Petri Dish 60 mm | Falcon BD | 351007 | |
| Picoinjector | XenoWorks Digital Microinjector | any standard model | |
| Pipette puller | Flaming-Brown Micropipette puller | Model P-1000 | |
| pIVT plasmid | AddGene | 32374 | Empty vector suitable for oocyte expression. |
| Pregnant Mare Serum Gonadotropin | Lee BioSolutions | 493-10 | |
| QIAprep Spin Miniprep Kit | Qiagen | 27104 | purification of up to 20 uL of plasmid DNA |
| QIAquick PCR purification kit | Qiagen | 28104 | |
| Quikchange II site directed mutagenesis kit | Agilent | 200523 | mutagenesis kit for insertions and deletions |
| Quikchange lightning multi-site directed mutagenesis kit | Agilent | 210512 | mutagenesis kit for single site changes |
| Scissors (Fine point) | Fine science tools | 14393 | |
| Scissors (Medium point) | Fine science tools | WP114225 | |
| Seal-Rite 1.5 mL microcentrifuge tube | USA Scientific | 1615-5500 | |
| Slide Warmer | any standard model | ||
| Spectrophotometer (Nanodrop) | Thermo Fisher Scientific | ND-ONE-W | |
| Stereomicroscope | any standard model | ||
| Subcloning Efficiency DH5a Competent Cells | Thermo Fisher Scientifc | 18265017 | |
| Syringe | BD Bioscienes | 309623 | 1 ml, 27G(1/2) |
| T4 DNA Ligase | New England Biolabs | M0202L | |
| T7 mMessage Machine high-yield capped RNA transcription kit | Life Technologies | AM1340 | |
| TritonX-100 | Sigma-Aldrich | x-100 | |
| Tween-20 | Sigma-Aldrich | 274348 | |
| Tweezer (Fine point- Size 5) | Fine science tools | SN.743.12.1 | |
| UltraPure Dnase/Rnase-Free Distilled Water | Thermo Fisher Scientifc | 10977015 | |
| UltraPure Ethidium Bromide 10mg/mL | Thermo Fisher Scientifc | 15585011 | |
| UVP UV/White lite transilluminator | Fisher Scientific | UV95041501 | |
| Vectashield Mounting Medium | Vector Laboratories | H-1000 |
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Cite This Article
Marin, D., Nguyen, A. L., Scott, Jr., R. T., Schindler, K. Using Mouse Oocytes to Assess Human Gene Function During Meiosis I. J. Vis. Exp. (134), e57442, doi:10.3791/57442 (2018).