Name: Fremstilling av kyllinglinse for mikroinjeksjon av et 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

Studerer proteinuttrykk under utvikling av kyllinglinser

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.

Forfatter Spotlight: Undersøkelse av linseutvikling og funksjon gjennom mikroinjeksjon av RCAS(A) retrovirus i embryonal kyllinglinse 

Mikroinjeksjon av rekombinant RCAS(A) retrovirus i embryonal kyllinglinse

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

Fremstilling av kyllinglinse for mikroinjeksjon av et RCAS(A) retrovirus

For å begynne, fest borosilikatglasskapillæren til gummipolstrede klips i den manuelle vertikale mikropipettavtrekkeren. Sett varmetemperaturen til mikropipettetrekkeren til 950 grader Celsius og utfør forhåndstrekking for å oppnå en tynnere og mykere glasskapillær. Sett deretter varmetemperaturen til 790 grader Celsius og utfør en sekundær trekking for å produsere endelige mikropipetter.Bruk en mikropipettekvern til å skjerpe spissåpningen forsiktig til en ytre diameter på 11 mikrometer. Legg deretter mikropipettene i en svampklemmepute inne i en glassburk. Inkubere de befruktede kyllingeggene til utviklingstrinn 18 i en 37 graders Celsius fuktet gyngende inkubator.Tørk av arbeidsområdet grundig med 70% etanol. Etter utvikling, fjern eggene fra inkubatoren. Spray eggene med 70% etanol og la dem lufttørke.Legg deretter egget på en eggholder med den større enden vendt oppover. Bruk et par skarpe tannede tang, bank forsiktig på den større enden av eggeskallet for å lage et hull på to centimeter i diameter. Fjern deretter fragmentene av eggeskallet.Plasser egget på et dissekerende mikroskopstadium og finn embryoet. Deretter bruker du den fine dissekerende tangen og saksen til å kutte av fosterhinnen som dekker toppen av embryoet. Legg deretter en 60 millimeter petriskål over eggets åpning.

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 ...