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

Tavuk Lensi Gelişimi Sırasında Protein Ekspresyonunun İncelenmesi

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.

Yazar Gündemi: Embriyonik Tavuk Lensinde RCAS(A) Retrovirüsünün Mikroenjeksiyonu Yoluyla Lens Gelişimi ve İşlevinin Araştırılması 

Rekombinant RCAS(A) Retrovirüsünün Embriyonik Tavuk Lensine Mikroenjeksiyonu

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

Bir RCAS(A) Retrovirüsünün Mikroenjeksiyonu için Tavuk Lensinin Hazırlanması

Başlamak için, borosilikat cam kılcalını manuel dikey mikropipet çektirmesindeki kauçuk yastıklı klipslere takın. Mikropipet çekicinin ısı sıcaklığını 950 santigrat dereceye ayarlayın ve daha ince ve yumuşak bir cam kılcal damar elde etmek için ön çekme işlemini gerçekleştirin. Ardından ısı sıcaklığını 790 santigrat dereceye ayarlayın ve son mikropipetleri üretmek için ikincil bir çekme işlemi gerçekleştirin.Bir mikropipet öğütücü kullanarak, uç açıklığını 11 mikrometre dış çapa kadar dikkatlice keskinleştirin. Ardından mikropipetleri bir cam kavanozun içindeki sünger sıkıştırma pedine yerleştirin. Döllenmiş tavuk yumurtalarını, 37 santigrat derece nemlendirilmiş sallanan inkübatörde gelişme aşaması 18'e kadar kuluçkaya yatırın.Çalışma alanını %70 etanol ile iyice silin. Geliştirmeden sonra, yumurtaları kuluçka makinesinden çıkarın. Yumurtalara %70 etanol püskürtün ve kurumaya bırakın.Ardından yumurtayı büyük ucu yukarı bakacak şekilde bir yumurta tutucuya yerleştirin. Bir çift keskin dişli forseps kullanarak, iki santimetre çapında bir delik oluşturmak için yumurta kabuğunun daha büyük ucuna dikkatlice vurun. Sonra yumurta kabuğunun parçalarını çıkarın.Yumurtayı diseksiyon mikroskobu aşamasına yerleştirin ve embriyoyu bulun. Daha sonra, embriyonun üstünü kaplayan amniyotik zarı kesmek için ince diseksiyon forseps ve makas kullanın. Ardından yumurtanın ağzının üzerine 60 milimetrelik bir Petri kabı yerleştirin.

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 Görüntüleme.  University of Texas Health Science Center, San Antonio. Başlamak için, borosilikat cam kılcalını manuel dikey mikropipet çektirmesindeki kauçuk yastıklı klipslere takın. Mikropipet çekicinin ısı sıcaklığını 950 santigrat dereceye ayarlayın ve daha ince ve yumuşak bir cam kılcal damar elde etmek için ön çekme işlemini gerçekleştirin. Ardından ısı sıcaklığını 790 santigrat dereceye ayarlayın ve son mikropipetleri üretmek için ikincil bir çekme işlemi gerçekleştirin.Bir mikropipet öğütücü kullanar...

Video: Bir RCAS(A) Retrovirüsünün Mikroenjeksiyonu için Tavuk Lensinin Hazırlanması

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 ...

Biyoloji

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.