microinjection of an rcas(a) retrovirus into embryonic chicken lens for expressing exogenous proteins 

研究鸡晶状体发育过程中的蛋白质表达

The goal of this protocol is to describe the methodology of embryonic chicken lens microinjection of an RCAS(A) (replication-competent avian sarcoma/leukosis retrovirus A). Effective retroviral delivery in an embryonic chicken lens has been demonstrated to be a promising tool for the in vivo study of the molecular mechanism and structure-function of lens proteins in normal lens physiology, pathological conditions, and development. Moreover, this experimental model could be used for the identification of therapeutic targets and drug screening for conditions such as human congenital cataracts. In all, this protocol aims to lay out the necessary steps for the development of a customizable platform for the study of lens proteins.
Embryonic chicks (Gallus domesticus), owing to their similarity in lens structure and function with the human lens, are a well-established animal model for the study of lens development and physiology1,2,3,4. The use of an RCAS(A) replication-competent avian retrovirus has been regarded as a highly effective, rapid, and customizable system to express exogenous proteins in chick embryos. Notably, it has a unique ability to confine the target gene transfer to proliferative lens fiber cells without the need for tissue-specific promoters, using the unique embryonic development time frame in which the presence of empty lens lumen permits in situ RCAS(A) microinjection into the restricted site for the expression of exogenous proteins within proliferative lens fiber cells5,6,7,8.
The chick embryo microinjection procedure, described in-depth here, is based originally partially on the work of Fekete et. al.6 and further developed by Jiang et. al.8 and has been utilized as a means of introducing both viral and nonviral plasmids into the lens of embryonic chicks1,9,10,11,12,13. Overall, the previous work demonstrates the potential of utilizing this methodology to study lens development, differentiation, cellular communication, and disease progression, and for the discovery and testing of therapeutic targets for lens pathological conditions such as cataracts.

作者聚焦：通过在胚胎鸡晶状体中显微注射 RCAS（A） 逆转录病毒来研究晶状体的发育和功能 

重组RCAS（A）逆转录病毒显微注射到胚胎鸡晶状体中

The presented methodology of embryonic chicken lens microinjection of a RCAS retrovirus is meant as a tool for studying in situ function and expression of proteins during lens development. Compared to other approaches, such as transgenic models and ex vivo cultures, the RCAS replication-competent avian retrovirus provides a highly-effective, rapid, and customizable system for expressing exogenous proteins in chick embryos. Targeted gene transfer can be confined to proliferative lens fiber cells without the need for promoters.There is prominent potential for utilizing this methodology to study lens development, differentiation, cellular communication, and disease progression, and for the discovery and testing of therapeutic targets for lens pathological conditions such as cataracts.

Watch this Scientific Journal Video about Microinjection of an RCASA Retrovirus into Embryonic Chicken Lens for Expressing Exogenous Proteins at JoVE.com

Microinjection of an RCAS(A) Retrovirus into Embryonic Chicken Lens for Expressing Exogenous Proteins   

将RCAS（A）逆转录病毒显微注射到胚胎鸡晶状体中以表达外源蛋白 

首先，在冰上解冻RCAS（A）逆转录病毒储备液。为避免堵塞微量移液器，将溶液以 10， 000 G 在 4 摄氏度下离心 10 秒，然后将上清液转移到新管中，同时将溶液保持在冰上。将拉伸的实验室石蜡膜放在 35 x 10 毫米的细胞培养皿上，并在石蜡膜中加入一微升无菌 PBS。将准备好的玻璃微量移液器连接到自动 Pico 进样器。按下填充模式将PBS填充到微量移液器中，然后按注射模式测试分配的一微升液体。将第二片拉伸的实验室封口膜放在新的细胞培养皿上，并在薄膜中加入一微升快速绿色染色的病毒储备液。将微量移液器的尖端降低到病毒储液器中，然后按下填充模式，用一微升病毒储液器填充微量移液器。在解剖显微镜下，将连接到自动注射器的填充微量移液管降低到鸡胚透镜的目标区域。然后调整光源以提供晶状体囊泡的清晰视图。在确保微量移液器位于晶状体腔内的正确位置后，注射 5 至 40 纳升病毒储备液。注射后，等待45秒，然后轻轻取出微量移液器。接下来，通过检查晶状体空腔中的快速绿色染料来检查显微注射是否成功，没有泄漏。检查后，用透明胶带密封蛋壳开口，并将鸡蛋放入 37 摄氏度加湿的静态培养箱中。本研究通过免疫荧光证明了嵌合连接子 50 和 43 环的组织学评估。使用Anti-Flag标记，将外源性连接子与内源性连接子区分开来。该研究还评估了连接子 50 的细胞内环域与水通道蛋白零点之间的相互作用。

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Research

JoVE Journal

Biology

Microinjection of an RCAS(A) Retrovirus into Embryonic Chicken Lens for Expressing Exogenous Proteins 

92 次观看.  University of Texas Health Science Center, San Antonio. 首先，在冰上解冻RCAS（A）逆转录病毒储备液。为避免堵塞微量移液器，将溶液以 10， 000 G 在 4 摄氏度下离心 10 秒，然后将上清液转移到新管中，同时将溶液保持在冰上。将拉伸的实验室石蜡膜放在 35 x 10 毫米的细胞培养皿上，并在石蜡膜中加入一微升无菌 PBS。将准备好的玻璃微量移液器连接到自动 Pico 进样器。按下填充模式将PBS填充到微量移液器中，然后按注射模?...

视频: Microinjection of an RCAS(A) Retrovirus into Embryonic Chicken Lens for Expressing Exogenous Proteins   

Microinjection of Recombinant RCAS(A) Retrovirus into Embryonic Chicken Lens

Embryonic chicken (Gallus domesticus) is a well-established animal model for the study of lens development and physiology, given its high degree of similarity with the human lens. RCAS(A) is a replication-competent chicken retrovirus that infects dividing cells, which serves as a powerful tool to study the in situ expression and function of wild-type and mutant proteins during lens development by microinjection into the empty lumen of lens vesicle at early developmental stages, restricting its action to surrounding proliferating lens cells. Compared to other approaches, such as transgenic models and ex vivo cultures, the use of an RCAS(A) replication-competent avian retrovirus provides a highly effective, rapid, and customizable system to express exogenous proteins in chick embryos. Specifically, targeted gene transfer can be confined to proliferative lens fiber cells without the need for tissue-specific promoters. In this article, we will briefly overview the steps needed for recombinant retrovirus RCAS(A) preparation, provide a detailed, comprehensive overview of the microinjection procedure, and provide sample results of the technique.

This protocol paper describes the methodology of embryonic chicken lens microinjection of an RCAS(A) retrovirus as a tool for studying in situ function and expression of proteins during lens development.

Embryonic chicken (Gallus domesticus) is a well-established animal model for the study of lens development and physiology, given its high degree of ...

科研

生物学

Preparation of Chicken Lens for Microinjection of an RCAS(A) Retrovirus

To begin, attach the borosilicate glass capillary to the rubber cushioned clips in the manual vertical micropipette puller. Set the heat temperature of the micropipette puller to 950 degrees Celsius and perform pre-pulling to obtain a thinner and softer glass capillary. Then set the heat temperature to 790 degrees Celsius and perform a secondary pulling to produce final micropipettes.Using a micropipette grinder, carefully sharpen the tip opening to an outer diameter of 11 micrometers. Then place the micropipettes in a sponge clamping pad inside of a glass jar. Incubate the fertilized chicken eggs until development stage 18 in a 37 degree Celsius humidified rocking incubator.Wipe down the working area thoroughly with 70%ethanol. After development, remove the eggs from the incubator. Spray the eggs with 70%ethanol and allow them to air dry.Then place the egg on an egg holder with the larger end facing upwards. Using a pair of sharp toothed forceps, carefully tap on the larger end of the eggshell to create a two centimeter diameter hole. Then remove the fragments of the egg shell.Place the egg on a dissecting microscope stage and locate the embryo. Next, use the fine dissecting forceps and scissors to cut off the amniotic membrane covering the top of the embryo. Then place a 60 millimeter Petri dish over the opening of the egg.

用于显微注射RCAS（A）逆转录病毒的鸡晶状体的制备

To begin, thaw the RCAS(A)retrovirus stock on ice. To avoid clogging the micropipette, centrifuge the solution at 10, 000 G for 10 seconds at four degrees Celsius and transfer the supernatant into a new tube while keeping the solutions on ice. Place a stretched laboratory parafilm on a 35 by 10 millimeter cell culture dish and add one microliter of sterile PBS to the parafilm.Connect a prepared glass micropipette to an automatic Pico injector. Press the fill mode to fill the PBS into a micropipette and then press the inject mode to test the allocated one microliter of liquid. Place a second piece of stretched laboratory parafilm on a new cell culture dish and add one microliter of fast green stained viral stock to the film.Lower the tip of the micropipette into the viral stock, and press the fill mode to fill the micropipette with one microliter of viral stock. Under a dissecting microscope, lower the filled micropipette connected to an automatic injector into the target region of the chicken embryo lens. Then adjust the light source to provide a clear view of the lens vesicle.After ensuring the micropipette is within the correct location inside the lens lumen, inject five to 40 nanoliters of the viral stock. After injection, wait 45 seconds before gently removing the micropipette. Next, check the successful microinjection by examining the fast green dye in the empty lumen of the lens without an leakage.Following examination, seal the eggshell opening with scotch tape and place the eggs in a 37 degree Celsius humidified static incubator. This study demonstrates the histological evaluation of the chimeric connexon 50 and 43 loops through immunofluorescence. Using Anti-Flag labeling, exogenous connexons were distinguished from endogenous ones.The study also evaluates the interaction between the intracellular loop domain of connexon 50 and Aquaporin zero.