小鼠 体内 胎盘靶向CRISPR操作
Summary
在这里，我们描述了一种时间特异性方法，以有效操纵 体内小鼠胎盘中的关键发育途径。这是通过在胚胎第12.5天将CRISPR质粒注射和电穿孔到妊娠母亲的胎盘中进行的。
Abstract
胎盘是调节和维持子宫 内哺乳动物发育的重要器官。胎盘负责母亲和胎儿之间营养物质和废物的转移以及生长因子和激素的产生和输送。小鼠胎盘遗传操作对于了解胎盘在产前发育中的特定作用至关重要。表达胎盘特异性Cre的转基因小鼠具有不同的有效性，其他胎盘基因操作方法可能是有用的替代方案。本文描述了一种利用CRISPR基因操作直接改变胎盘基因表达的技术，可用于修饰靶基因的表达。使用相对先进的手术方法，怀孕的母亲在胚胎第12.5天（E12.5）进行剖腹手术，并通过玻璃微量移液器将CRISPR质粒输送到单个胎盘中。质粒在每次进样后立即电穿孔。母体恢复后，胎盘和胚胎可以继续发育，直到稍后的时间点进行评估。使用该技术后对胎盘和后代的评估可以确定时间特异性胎盘功能在发育中的作用。这种类型的操作将允许更好地了解胎盘遗传学和功能如何影响多种疾病背景下的胎儿生长和发育。
Introduction
胎盘是参与胎儿发育的重要器官。胎盘的主要作用是提供基本因素并调节营养物质和废物与胎儿之间的转移。哺乳动物胎盘由胎儿和母体组织组成，它们构成了胎母界面，因此，母体和胎儿的遗传学影响功能1。胎盘的遗传异常或功能受损会极大地改变胎儿的发育。先前的工作表明，胎盘遗传学和发育与胎儿特定器官系统的发育改变有关。特别是，胎盘异常与胎儿大脑，心脏和血管系统的变化有关2，3，4，5。
激素、生长因子和其他分子从胎盘到胎儿的运输在胎儿发育中起着重要作用6。已经表明，改变特定分子的胎盘产生可以改变神经发育。母体炎症可以通过改变胎盘中的色氨酸（TRP）代谢基因表达来增加血清素的产生，从而在胎儿大脑中产生血清素7。其他研究发现胎盘异常与心脏缺陷。胎盘异常被认为会导致先天性心脏缺陷，这是人类最常见的出生缺陷8。最近的一项研究已经确定了几个在胎盘和心脏中具有相似细胞途径的基因。如果被破坏，这些途径可能会导致两个器官的缺陷9。胎盘缺陷可能会加剧先天性心脏缺陷。胎盘遗传学和功能对特定胎儿器官系统发育的作用是一个新兴的研究领域。
小鼠具有血绒毛膜胎盘和人类胎盘的其他特征，这使它们成为研究人类疾病的非常有用的模型1。尽管胎盘很重要，但目前缺乏有针对性的体内基因操作。此外，目前有更多的选择可用于敲除或敲低，而不是胎盘中的过表达或功能获得操作10。有几种表达Cre的转基因系用于胎盘特异性操作，每种系在不同的时间点位于不同的滋养层谱系中。这些包括Cyp19-Cre，Ada/Tpbpa-Cre，PDGFRα-CreER和Gcm1-Cre 11，12，13，14。虽然这些Cre转基因是有效的，但它们可能无法在特定的时间点操纵某些基因。敲除或过表达胎盘基因表达的另一种常用方法是将慢病毒载体插入囊胚培养物中，这会导致滋养层特异性遗传操作15，16。该技术允许胎盘基因表达在发育早期发生强劲变化。RNA干扰在体内的使用在胎盘中很少使用。shRNA质粒的插入可以类似于本文中描述的CRIPSR技术进行。这已在E13.5完成，以成功降低胎盘中的PlGF表达，对后代脑脉管系统产生影响17。
除了主要用于敲除或敲低的技术外，诱导过表达通常使用腺病毒或插入外源性蛋白进行。用于过表达的技术具有不同的成功率，并且大多在妊娠后期进行。为了研究胰岛素样生长因子1（IGF-1）在胎盘功能中的作用，进行了腺病毒介导的胎盘基因转移以诱导IGF-1基因18，19的过表达。这是在小鼠妊娠后期通过直接胎盘注射在E18.5上进行的。为了提供额外的选择并规避已建立的胎盘遗传操作的可能失败，例如Cre-Lox组合失败，腺病毒的可能毒性以及shRNA的脱靶效应，可以使用胎盘的体内直接CRISPR操作20，21，22。开发该模型是为了解决缺乏过表达模型的问题，并创建一个具有灵活性的模型。
该技术基于Lecuyer等人的工作，其中shRNA和CRISPR质粒在体内直接靶向小鼠胎盘以改变PlGF表达17。该技术可用于在多个时间点使用CRISPR操作直接改变胎盘基因表达;对于这项工作，选择了E12.5。此时胎盘已经成熟，并且足够大，可以操纵，允许在E12.5上插入特定的CRISPR质粒，这可能对怀孕中晚期的胎儿发育产生重大影响23，24。与转基因方法不同，但与病毒诱导或RNA干扰类似，该技术允许使用相对先进的手术方法在特定时间点进行过表达或敲除，从而避免早期变化可能造成的胎盘受损或胚胎致死性。由于只有少数胎盘在一窝中接受实验质粒或对照质粒，因此该方法允许两种类型的内部对照。这些质控品是用适当的对照质粒进样和电穿孔的质粒，以及未接受直接操作的质粒。该技术经过优化，通过协同激活介质（SAM）CRISPR质粒在小鼠胎盘中产生IGF-1基因的过表达。选择了 IGF-1 基因，因为 IGF-1 是一种主要的生长激素，主要在出生前在胎盘中产生25，26.这种新的胎盘靶向CRISPR技术将允许直接操作，以帮助确定胎盘功能与胎儿发育之间的联系。
Protocol
所有程序均按照联邦法规和爱荷华大学政策执行，并得到机构动物护理和使用委员会的批准。 1. 畜牧业 将动物保持在12小时的日光循环 中，随意进食和水。 使用8-15周龄的CD-1雌性小鼠。使用交配插头的存在来识别 E0.5。 在E0.5上，单独容纳怀孕的水坝。 2. 微量移液器的校准 注…
Representative Results
一般手术结果（图6）在这项研究中，有三个纵的组。这些包括注射普通CRISPR Cas9对照质粒（Cas9对照），活化对照CRISPR质粒（行动对照）或IGF-1 SAM活化质粒（Igf1-OE）的胎盘。Cas9对照更适合敲除质粒，活化对照更适合过表达/活化质粒。为了评估通过注射和电穿孔操纵胎盘引起的活力变化，在E14.5上分析了垫料中的胚胎存活率（<strong cla…
Discussion
胎盘是胎儿生长的主要调节因子，如前所述，胎盘基因表达或功能的变化可能会显着影响胎儿发育6。这里概述的方案可用于使用相对先进的手术方法对小鼠胎盘进行靶向体内CRISPR操作。该技术允许大量可存活的胚胎及其相应的胎盘，可用于进一步研究（图6A，B）。这项技术使我们能够成功地在E14.5上过表达胎盘IGF-1（<strong class="xfi…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者承认以下资金来源：R01 MH122435，NIH T32GM008629和NIH T32GM145441。作者感谢Val Sheffield博士和Calvin Carter博士在爱荷华大学的实验室使用他们的手术室和设备，以及Eric Van Otterloo博士，Nandakumar Narayanan博士和Matthew Weber博士在显微镜方面的帮助。作者还感谢Sara Maurer博士，Maya Evans和Sreelekha Kundu对试点手术的帮助。
Materials
| 1.5 ml Tubes | USA Scientific Inc | 1615-5500 | |
| 4% Paraformeldhyde (PFA) in PBS | Thermo Fisher Scientific | J61899.AP | |
| 96 Well plate | Cornings | 3598 | For BCA kit |
| Absorbent Underpads | Fisher Scientific | 14-206-62 | |
| Activation Control Plasmid | Santa Cruz Biotechnology | sc-437275 | Dnase-free water provided for dilution |
| AMV Reverse Transcriptase | New England Biolabs | M0277L | Use for cDNA synthesis |
| Anesthetic Gas Vaporizor | Vetamac | VAD-601TT | VAD-compact vaporizer |
| Artifical Tear Gel | Akorn | NDC 59399-162-35 | |
| BCA Protein Assay Kit | Thermo Fisher Scientific | 23227 | Protein quantification |
| Biovortexer | Bellco Glass, Inc. | 198050000 | Hand-held tissue homogenizer |
| CellSens Software | Olympus | V4.1.1 | Image processing to FISH images. |
| Centrifuge 5810 | Eppendorf | EP022628168 | Plate centrifuge |
| Chloroform | Thermo Fisher Scientific | J67241-AP | RNA isolation |
| Cotton Tipped Applicators | ProAdvantage | 77100 | Sterilize before use |
| CRISPR/Cas9 Control Plasmid | Santa Cruz Biotechnology | sc-418922 | Dnase-free water provided for dilution |
| CryoStat | Leica | CM1950 | |
| Dissection Microscope | Leica | M125 C | Used for post-necroscopy imaging |
| Dissolvable Sutures | Med Vet International | J385H | |
| Distilled Water | Gibco | 15230162 | |
| Dulbecco's Phosphate Buffered Saline (DPBS) | Thermo fisher Scientific | 14190144 | (-) Calcium; (-) Magnesium |
| ECM 830 Electro Electroporator (Electroporation Machine) | BTX Harvard Apparatus | 45-0662 | Generator only |
| Electric Razor | Wahl | CL9990 | Kent Scientific |
| Electroporation paddles/Tweezertrodes | BTX Harvard Apparatus | 45-0487 | 3 mm diameter paddles; wires included |
| Embedding Cassette: 250 PK | Grainger | 21RK94 | Placenta embedding cassettes |
| Ethanol | Thermo Fisher Scientific | 268280010 | |
| F-Air Canisters | Penn Veterinary Supply Inc | BIC80120 | Excess isoflurane filter |
| Fast Green Dye FCF | Sigma | F7252-5G | Dissolve to 1 μg/ml and filter; protect from light |
| Filter-based microplate photometer (plate reader) | Fisher Scientific | 14377576 | Can be used for BCA and ELISA |
| Forceps | VWR | 82027-386 | Fine tips, straight, serrated |
| Formalin solution, neutral buffered, 10% | Sigma Aldrich | HT501128 | |
| Glass Capillaries – Borosilicate Glass (Micropipette) | Sutter Instrument | B150-86-10 | O.D.: 1.5 mm, I.D.: 0.86 mm, 10 cm length |
| Halt Protease and Phosphotase inhibitor cocktail (100x) | Thermo Scientific | 1861281 | Protein homogenization buffer |
| Heating Pad | Thermotech | S766D | Digitial Moist Heating Pad |
| Hemostats | VWR | 10806-188 | Fully surrated jaw; curved |
| Hot Water Bath | Fisher Scientific | 20253 | Isotemp 205 |
| Igf-1 SAM Plasmid (m1) | Santa Cruz Biotechnology | sc-421056-ACT | Dnase-free water provided for dilution |
| Induction Chamber | Vetamac | 941443 | No specific liter size required |
| Isoflurane | Piramal Pharma Limited | NDC 66794-013-25 | |
| Isoproponal/2-Proponal | Fisher Scientific | A451-4 | RNA isolation |
| Ketamine HCl 100mg/ml | Akorn | NDC 59399-114-10 | |
| MgCl2/Magneisum Chloride | Sigma Aldrich | 63069-100ML | 1M. Protein homogenization buffer |
| MicroAmp™ Optical 384-Well Reaction Plate with Barcode | Fisher Scientific | 4309849 | Barcoded plates not required |
| Microcapillary Tip | Eppendorf | 5196082001 | Attached to BTX Microinjector |
| Microinjector | BTX Harvard Apparatus | 45-0766 | Stainless Steel Pipette Holder, 130 mm Length, for 1 to 1.5 mm Pipettes |
| Microject 1000A (Injection Machine) | BTX Harvard Apparatus | 45-0751 | MicroJect 1000A Plus System |
| Micropipette Puller Model P-97 | Sutter Instrument | P-97 | Flaming/Brown type micropipette puller |
| Microplate Mixer (Plate Shaker) | scilogex | 822000049999 | |
| Mouse/Rat IGF-I/IGF-1 Quantikine ELISA Kit | R & D Systems | MG100 | |
| Needles | BD – Becton, Dickson, and Company | 305106 | 30 Gx 1/2 (0.3 mm x 13 mm) |
| Nitrogen Tank | Linde | 7727-37-9 | Any innert gas |
| Non-Steroidal Anti-Inflammatory Drug (NSAID) | Norbrook Laboratories Limited | NDC 55529-040-10 | Analesgic such as Meloxicam |
| Nose Cone | Vetamac | 921609 | 9-14 mm |
| Opal 620 detection dye | Akoya Biosciences | SKU FP1495001KT | Used for FISH |
| Optimal Cutting Temperature (O.C.T) Compound | Sakura | 4583 | |
| Oxygen Tank | Linde | 7782 – 44 – 7 | Medical grade oxygen |
| Pestles | USA Scientific Inc | 14155390 | |
| Povidone-Iodine Solution, 5% | Avrio Health L.P. | NDC 67618-155-16 | |
| Power SYBR™ Green PCR Master Mix | Thermo Fisher Scientific | 4367659 | Use for qPCR |
| Random Hexamers (Random Primers) | New England Biolabs | S1330S | Use for cDNA synthesis |
| Razor Blade | Grainger | 26X080 | |
| RNA Cleanup Kit & Concentrator | Zymo Research | R1013 | |
| RNALater | Thermo Fisher Scientific | AM7021 | |
| RNAscope kit v.2.5 | Advanced Cells Diagnostics | 323100 | Contains all reagents required for fluorescent in situ hybridization. Probes sold separately. |
| RNAscope™ Probe- Mm-Prl8a8-C2 | Advanced Cells Diagnostics | 528641-C2 | |
| RNAscope™ Probe- Vector-dCas9-3xNLS-VP64 | Advanced Cells Diagnostics | 527421 | |
| Roto-Therm Mini | Benchmark | R2020 | Dry oven for in situ hybridization |
| Scissors | VWR | 82027-578 | Dissecting Scissors, Sharp Tip, 4¹/₂ |
| Sodium Chloride (Saline) | Hospra | NDC 0409-4888-03 | Sterile, 0.9% |
| Sodium Citrate, Trisodium Salt, Dihydrate, [Citric Acid, Trisodium Dihydrate] | Research Product International | 03-04-6132 | |
| Sodium Hydroxide 1N Concentrate, Fisher Chemical | Fisher Scientific | SS277 | Protein homogenization buffer |
| Steamer | Bella | B00DPX8UBA | |
| Sterile Surgical Drape | Busse | 696 | Sterilize before use |
| Superfrost Plus Microscope Slides | Fisher Scientific | 12-550-15 | |
| Surgipath Cover Glass 24×60 | Leica | 3800160 | |
| Syringes | BD – Becton, Dickson, and Company | 309659 | BD Luer Slip Tip Syringe sterile, single use, 1 mL |
| Thermo Scientific™ Invitrogen™ Nanodrop™ One Spectrophotometer with WiFi and Qubit™ 4 Fluorometer | Fisher Scientific | 13-400-525 | This configuration comes with Qubit 4 fluorometer. Qubit quantification not required. |
| Tissue Adhesive | 3M | 1469SB | VetBond |
| Tris HCl | Thermo Fisher Scientific | 15568025 | 1M. Protein homogenization buffer |
| TRIzol™ Reagent | Thermo Fisher Scientific | 15596018 | RNA isolation |
| TSA Buffer Pack | Advanced Cells Diagnostics | 322810 | Used to dilute Opal 620 detection dye |
| Universal F-Circuit | Vetamac | 40200 | Attached to vaporizer and vaporizer accessories |
| Upright Compound Fluorescence Microscope | Olympus | BX61VS | Used for FISH imaging |
| Vectorshield with DAPI | Vector Laboratories | H-1200 | Coverslip mounting media |
| ViiA™ 7 Real-Time PCR System with 384-Well Block | Thermo Fisher Scientific | 4453536 | This is for SYBR 384-well block detection. TaqMan and/or smaller blocks available |
| Wet n Wild Nail Polish Wild Shine, Clear Nail Protector, Nail Color | Amazon | C450B | |
| Xylazine 20mg/ml | Anased | 343730_RX |
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Cite This Article
Carver, A. J., Taylor, R. J., Stevens, H. E. Mouse In Vivo Placental Targeted CRISPR Manipulation. J. Vis. Exp. (194), e64760, doi:10.3791/64760 (2023).