Name: Beredning av kycklinglins för mikroinjektion av ett RCAS(A)-retrovirus
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

Studerar proteinuttryck under utveckling av kycklinglins

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.

Författare Spotlight: Undersöker linsutveckling och funktion genom mikroinjektion av RCAS(A) retrovirus i embryonal kycklinglins 

Mikroinjektion av rekombinant RCAS(A)-retrovirus i embryonal kycklinglins

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

Beredning av kycklinglins för mikroinjektion av ett RCAS(A)-retrovirus

Till att börja med, fäst bosilikatglaskapillären på de gummidämpade klämmorna i den manuella vertikala mikropipettavdragaren. Ställ in mikropipettavdragarens värmetemperatur på 950 grader Celsius och utför fördragning för att få en tunnare och mjukare glaskapillär. Ställ sedan in värmetemperaturen på 790 grader Celsius och utför en sekundär dragning för att producera slutliga mikropipetter.Använd en mikropipettkvarn och vässa försiktigt spetsöppningen till en ytterdiameter på 11 mikrometer. Placera sedan mikropipetterna i en svampklämdyna inuti en glasburk. Ruva de befruktade hönsäggen fram till utvecklingsstadium 18 i en 37 grader Celsius fuktad gunginkubator.Torka av arbetsområdet noggrant med 70 % etanol. Efter utvecklingen tar du bort äggen från inkubatorn. Spraya äggen med 70% etanol och låt dem lufttorka.Lägg sedan ägget på en ägghållare med den större änden uppåt. Använd en vass pincett och knacka försiktigt på den större änden av äggskalet för att skapa ett hål med en diameter på två centimeter. Ta sedan bort fragmenten av äggskalet.Placera ägget i ett dissekerande mikroskop och lokalisera embryot. Använd sedan den fina dissekerande pincetten och saxen för att klippa av fosterhinnan som täcker toppen av embryot. Lägg sedan en 60 millimeters petriskål över äggets öppning.

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 ...