重组RCAS（A）逆转录病毒显微注射到胚胎鸡晶状体中
Summary
该协议论文描述了RCAS（A）逆转录病毒的胚胎鸡晶状体显微注射的方法，作为研究晶状体发育过程中蛋白质原 位 功能和表达的工具。
Abstract
胚胎鸡（Gallus domesticus）是一种成熟的动物模型，用于研究晶状体发育和生理学，因为它与人类晶状体高度相似。RCAS（A）是一种具有复制能力的鸡逆转录病毒，可感染分裂细胞，是研究晶状体发育过程中野生型和突变蛋白原 位 表达和功能的有力工具，在早期发育阶段通过显微注射到晶状体囊泡的空腔中，将其作用限制在周围增殖的晶状体细胞中。与转基因模型和 离体 培养等其他方法相比，使用具有复制能力的禽逆转录病毒提供了一种高效、快速和可定制的系统来表达雏鸡胚胎中的外源蛋白。具体来说，靶向基因转移可以局限于增殖的晶状体纤维细胞，而不需要组织特异性启动子。在本文中，我们将简要概述重组逆转录病毒RCAS（A）制备所需的步骤，提供显微注射程序的详细、全面的概述，并提供该技术的样本结果。
Introduction
该协议的目的是描述RCAS（A）（具有复制能力的禽肉瘤/白血病逆转录病毒A）的胚胎鸡晶状体显微注射的方法。在胚胎鸡晶状体中有效的逆转录病毒递送已被证明是 体内研究晶 状体蛋白在正常晶状体生理学、病理状况和发育中的分子机制和结构功能的有前途的工具。此外，该实验模型还可用于人类先天性白内障等疾病的治疗靶点识别和药物筛选。总而言之，该协议旨在为开发用于研究晶状体蛋白的可定制平台制定必要的步骤。
胚胎雏鸡（Gallus domesticus）由于其晶状体结构和功能与人类晶状体相似，是研究晶状体发育和生理学的成熟动物模型1,2,3,4。使用具有复制能力的 RCAS（A） 禽逆转录病毒被认为是在雏鸡胚胎中表达外源蛋白的高效、快速和可定制的系统。值得注意的是，它具有独特的能力，可以将靶基因转移限制在增殖的晶状体纤维细胞中，而不需要组织特异性启动子，使用独特的胚胎发育时间框架，其中空晶状体腔的存在允许原位 RCAS（A） 显微注射到受限位点，以便在增殖晶状体纤维细胞内表达外源性蛋白质5，6,7,8.
这里深入描述的鸡胚显微注射程序最初部分基于 Fekete 等人的工作。al.6 并由 Jiang 等人进一步发展。Al.8，并已被用作将病毒和非病毒质粒引入胚胎雏鸡晶状体的手段1,9,10,11,12,13。总体而言，先前的工作证明了利用这种方法研究晶状体发育、分化、细胞通讯和疾病进展的潜力，以及发现和测试晶状体病理状况（如白内障）的治疗靶点。
Protocol
本研究是按照《动物福利法》和《动物福利实施条例》的原则，根据《实验动物护理和使用指南》的原则进行的。所有动物程序均由德克萨斯大学圣安东尼奥健康科学中心机构动物护理和使用委员会批准。有关协议的概述，请参阅 图 1;有关本协议中使用的所有材料、试剂和仪器的详细信息，请参阅 材料表 。 <p class="jove_content biglegend" fo:keep-together.within-page="1"…
Representative Results
在确定特定靶蛋白和鉴定相关基因序列后，总体实验方法包括将基因序列克隆到逆转录病毒 RCAS（A） 载体中，方法是初始克隆到接头载体中，然后是病毒载体。其次，使用包装细胞制备高滴度病毒颗粒，以收获和浓缩病毒粒子。前两个主要步骤已基本描述，代表性结果在其他地方呈现6、7、8、14、<sup…
Discussion
该实验模型提供了在完整晶状体中表达目标蛋白质的机会，从而研究了这些蛋白质在晶状体结构和功能中的功能相关性。胚胎雏鸡显微注射模型部分基于Fekete等人的工作。al.6，并由Jiang等人进一步发展。al.8 并已被用作将病毒质粒和试剂（如激动剂、小干扰 RNA （siRNA） 和肽）插入雏鸡晶状体的手段 1,9,10,11,12,13。<…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了美国国立卫生研究院（NIH）资助：RO1 EY012085（给JXJ）和F32DK134051（给FMA）以及韦尔奇基金会资助：AQ-1507（给JXJ）。内容完全由作者负责，并不一定代表美国国立卫生研究院的官方观点。这些数字部分是用 Biorender.com 创建的。
Materials
| 0.22 µm Filter | Corning | 431118 | For removing cellular debris from media |
| 35 mm x 10 mm Culture Dish | FisherScientific | 50-202-030 | For using during microinjection |
| Centrifuge | Fisherbrand | 13-100-676 | Spinning down solution |
| Constructs | GENEWIZ | – | For generation of constructs |
| Dissecting microscope | AmScope | SM-4TZ-144A | Visualization of lens for microinjection |
| DNA PCR primers | Integrated DNA Technologies | – | Generation of primers: Intracellular loop (IL)-deleted Cx50 (residues 1–97 and 149–400) as well as the Cla12NCO vector were obtained with the following pair of primers: sense, CTCCTGAGAACCTACATCCT; antisense, CACCGCATGCCCAAAGTACAC ILs of Cx43 (residues 98–150) and Cx46 (residues 98–166) were obtained with the following pairs of primers: sense, TACGTGATGAGGAAAGAAGAG; antisense, TCCTCCACGCATCTTTACCTTG; sense, CACATTGTACGCATGGAAGAG; antisense, AGCACCTCCC AT ACGGATTC, respectively Cla12NCO-Cx43 construct template was obtained with the following pair of primers: sense, CTGCTTCGTACTTACATCATC; antisense, GAACAC GTGCGCCAGGTAC ILs of Cx50 (residues 98–148) or Cx46 (residues 98–166) were cloned by using Cla12NCO-Cx50 and Cla12NCO-Cx46 constructs as the templates with the following pair of primers: sense, CACCATGTCCGCATGGAGGAGA; antisense, GGTCCCC TC CAGGCGAAAC; sense, CACATTGTACGCATGGAAGAG; antisense, AGCACCTCCCATACGGATTC, respectively |
| Drummond Nanoject II Automatic Nanoliter Injector | Drummond Scientific | 3-000-204 | Microinjection Pipet |
| Dual Gooseneck Lights Microscope Illuminator | AmScope | LED-50WY | Lighting for visualization |
| Dulbecco’s Modified Eagle Medium (DMEM) | Invitrogen | For cell culture | |
| Egg Holder | – | – | Homemade styrofoam rings with 2-inch diameter and one-half inch height |
| Egg Incubator | GQF Manufacturing Company Inc. | 1502 | For incubation of fertilized eggs |
| Fast Green | Fisher scientific | F99-10 | For visualization of viral stock injection |
| Fertilized white leghorn chicken eggs | Texas A&M University | N/A | Animal model of choice for microinjection (https://posc.tamu.edu/fertile-egg-orders/) |
| Fetal Bovine Serum (FBS) | Hyclone Laboratories | For cell culture | |
| Fluorescein-conjugated anti-mouse IgG | Jackson ImmunoResearch | 115-095-003 | For anti-FLAG 1:500 |
| Forceps | FisherScientific | 22-327379 | For moving things around and isolation |
| Glass capillaries | Sutter Instruments | B100-75-10 | Glass micropipette for microinjection (O.D. 1.0 mm, I.D. 0.75 mm, 10 cm length) |
| Lipofectamine | Invitrogen | L3000001 | For transfection |
| Manual vertical micropipette puller | Sutter Instruments | P-30 | To obtain glass micropipette of the correct size |
| Microcentrifuge Tubes | FisherScientific | 02-682-004 | Dissolving solution |
| Microscope | Keyence | BZ-X710 | For imaging staining |
| Parafilm | FisherScientific | 03-448-254 | Placing solution |
| Penicillin/Streptomycin | Invitrogen | For cell culture | |
| Pico-Injector | Harvard Apparatus | PLI-100 | For delivering small liquid volumes precisely through micropipettes by applying a regulated pressure for a digitally set period of time |
| rabbit anti-chick AQP0 | Self generated | – | Jiang JX, White TW, Goodenough DA, Paul DL. Molecular cloning and functional characterization of chick lens fiber connexin 45.6. Mol Biol Cell. 1994 Mar;5(3):363-73. doi: 10.1091/mbc.5.3.363. |
| rabbit anti-FLAG antibody | Rockland Immunichemicals | 600-401-383 | For staining FLAG |
| Rhodamine-conjugated anti-rabbit IgG | Jackson ImmunoResearch | 111-295-003 | For anti-AQP0 1:500 |
| Sponge clamping pad | Sutter Instruments | BX10 | For storage of glass micropipette |
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Cite This Article
Acosta, F. M., Ma, B., Gu, S., Jiang, J. X. Microinjection of Recombinant RCAS(A) Retrovirus into Embryonic Chicken Lens. J. Vis. Exp. (199), e65727, doi:10.3791/65727 (2023).