Name: Preparação de lente de frango para microinjeção de um retrovírus RCAS(A)
Uploaded: 2023-09-01T14:05:00
Description: Watch this Scientific Journal Video about Preparation of Chicken Lens for Microinjection of an RCASA Retrovirus at JoVE.com

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

Estudando a expressão de proteínas durante o desenvolvimento de lentes de galinha

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.

Autor em destaque: investigando o desenvolvimento e a função do cristalino por meio da microinjeção de retrovírus RCAS (A) em lentes embrionárias de galinha 

Microinjeção de Retrovírus RCAS(A) Recombinante em Lentes de Galinha Embrionárias

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

Preparação de lente de frango para microinjeção de um retrovírus RCAS(A)

Para começar, fixe o capilar de vidro borossilicato aos grampos de borracha almofadados no puxador manual vertical de micropipetas. Ajuste a temperatura térmica do extrator de micropipeta para 950 graus Celsius e realize a pré-tração para obter um capilar de vidro mais fino e macio. Em seguida, ajuste a temperatura do calor para 790 graus Celsius e execute uma tração secundária para produzir micropipetas finais.Usando um moedor de micropipeta, afie cuidadosamente a abertura da ponta para um diâmetro externo de 11 micrômetros. Em seguida, coloque as micropipetas em uma almofada de fixação de esponja dentro de um frasco de vidro. Incubar os ovos de galinha fertilizados até o estágio de desenvolvimento 18 em uma incubadora de balanço umidificada de 37 graus Celsius.Limpe bem a área de trabalho com etanol 70%. Após o desenvolvimento, retire os ovos da incubadora. Borrife os ovos com etanol 70% e deixe secar ao ar.Em seguida, coloque o ovo em um suporte de ovos com a extremidade maior voltada para cima. Usando um par de pinças dentadas afiadas, bata cuidadosamente na extremidade maior da casca do ovo para criar um furo de dois centímetros de diâmetro. Em seguida, remova os fragmentos da casca do ovo.Coloque o ovo em um estágio de microscópio dissecante e localize o embrião. Em seguida, use a pinça dissecante fina e a tesoura para cortar a membrana amniótica que cobre a parte superior do embrião. Em seguida, coloque uma placa de Petri de 60 milímetros sobre a abertura do ovo.

Research

JoVE Journal

Biology

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

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 ...