Name: Preparación de lente de pollo para la microinyección de un retrovirus 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

Estudio de la expresión de proteínas durante el desarrollo del cristalino de los pollos

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 destacado: Investigación del desarrollo y la función del cristalino mediante la microinyección de retrovirus RCAS(A) en el cristalino de pollo embrionario 

Microinyección de retrovirus RCAS(A) recombinante en lentes embrionarias de pollo

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

Preparación de lente de pollo para la microinyección de un retrovirus RCAS(A)

Para empezar, fije el capilar de vidrio de borosilicato a los clips acolchados de goma en el extractor de micropipetas vertical manual. Ajuste la temperatura de calor del extractor de micropipetas a 950 grados centígrados y realice la extracción previa para obtener un capilar de vidrio más delgado y blando. A continuación, ajuste la temperatura de calor a 790 grados centígrados y realice un tirón secundario para producir las micropipetas finales.Con una amoladora de micropipetas, afile con cuidado la abertura de la punta hasta un diámetro exterior de 11 micrómetros. A continuación, coloque las micropipetas en una almohadilla de sujeción de esponja dentro de un frasco de vidrio. Incubar los huevos de gallina fertilizados hasta la etapa de desarrollo 18 en una incubadora mecedora humidificada a 37 grados centígrados.Limpie bien el área de trabajo con etanol al 70%. Después del desarrollo, retire los huevos de la incubadora. Rocíe los huevos con etanol al 70% y déjelos secar al aire.A continuación, coloca el huevo en un portahuevos con el extremo más grande hacia arriba. Con un par de pinzas dentadas afiladas, golpee con cuidado el extremo más grande de la cáscara de huevo para crear un orificio de dos centímetros de diámetro. Luego retire los fragmentos de la cáscara del huevo.Coloque el óvulo en una platina de microscopio de disección y localice el embrión. A continuación, utiliza las finas pinzas de disección y las tijeras para cortar la membrana amniótica que cubre la parte superior del embrión. A continuación, coloca una placa de Petri de 60 milímetros sobre la abertura del huevo.

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 ...