preparation of chicken lens for microinjection of an rcas(a) retrovirus

닭 수정체 발달 중 단백질 발현 연구

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.

저자 스포트라이트: Investigating Lens Development and Function Through Microinjection of RCAS(A) Retrovirus in Embryonic Chicken Lens(배아 닭 수정체에서 RCAS(A) 레트로바이러스의 미세주입을 통한 렌즈 발달 및 기능 조사) 

재조합 RCAS(A) 레트로바이러스를 배아 닭 수정체에 미세주입

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 Preparation of Chicken Lens for Microinjection of an RCASA Retrovirus at JoVE.com

RCAS(A) 레트로바이러스의 미세주입을 위한 치킨 렌즈의 제조

시작하려면 붕규산 유리 모세관을 수동 수직 마이크로피펫 풀러의 고무 쿠션 클립에 부착합니다. 마이크로피펫 풀러의 열 온도를 섭씨 950도로 설정하고 사전 당김을 수행하여 더 얇고 부드러운 유리 모세관을 얻습니다. 그런 다음 가열 온도를 섭씨 790도로 설정하고 2차 당김을 수행하여 최종 마이크로피펫을 생성합니다.마이크로피펫 그라인더를 사용하여 팁 개구부를 외경 11마이크로미터로 조심스럽게 날카롭게 합니다. 그런 다음 마이크로피펫을 유리병 내부의 스폰지 클램핑 패드에 넣습니다. 수정된 닭고기 알을 섭씨 18도의 가습 흔들 인큐베이터에서 발달 단계 37까지 배양합니다.70% 에탄올로 작업 영역을 철저히 닦으십시오. 발달 후 인큐베이터에서 알을 꺼냅니다. 계란에 70% 에탄올을 뿌리고 자연 건조시킵니다.그런 다음 큰 쪽이 위를 향하도록 계란 홀더에 계란을 놓습니다. 날카로운 톱니가 있는 집게를 사용하여 달걀 껍질의 큰 쪽 끝을 조심스럽게 두드려 지름 2cm의 구멍을 만듭니다. 그런 다음 달걀 껍질 조각을 제거하십시오.계란을 해부 현미경 스테이지에 놓고 배아를 찾습니다. 그런 다음 미세한 해부 겸자와 가위를 사용하여 배아의 상단을 덮고 있는 양막을 잘라냅니다. 그런 다음 달걀 입구 위에 60mm 페트리 접시를 놓습니다.

preparation-of-chicken-lens-for-microinjection-of-an-rcasa-retrovirus

Research

JoVE Journal

Biology

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

67 조회수.  University of Texas Health Science Center, San Antonio. 시작하려면 붕규산 유리 모세관을 수동 수직 마이크로피펫 풀러의 고무 쿠션 클립에 부착합니다. 마이크로피펫 풀러의 열 온도를 섭씨 950도로 설정하고 사전 당김을 수행하여 더 얇고 부드러운 유리 모세관을 얻습니다. 그런 다음 가열 온도를 섭씨 790도로 설정하고 2차 당김을 수행하여 최종 마이크로피펫을 생성합니다.마이크로피펫 그라인더를 사용하여 팁 개구부를 ...

비디오: RCAS(A) 레트로바이러스의 미세주입을 위한 치킨 렌즈의 제조

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 ...

연구

생물학

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.

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

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.