Published: May 18th, 2022
We have developed a novel loss-of-function approach that involves the introduction and genomic integration of artificial micro-RNA sequences into chick embryos by using in ovo electroporation and the Tol2 transposon system. This technique provides a robust and stable gene knockdown methodology for studies of gene function during development.
The chick retina has long been an important model system in developmental neurobiology, with advantages including its large size, rapid development, and accessibility for visualization and experimental manipulations. However, its major technical limitation had been the lack of robust loss-of-function approaches for gene function analyses. This protocol describes a methodology of gene silencing in the developing chick retina that involves transgenic expression of artificial microRNAs (miRNAs) by using the Tol2 transposon system. In this approach, a Tol2 transposon plasmid that contains an expression cassette for the EmGFP (emerald green fluorescent protein) marker and artificial pre-miRNA sequences against a target gene is introduced into the embryonic chick retina with a Tol2 transposase expression construct by in ovo electroporation. In the transfected retinal cells, the transposase catalyzes the excision of the expression cassette from the transposon vector and its integration into host chromosomes, leading to the stable expression of miRNAs and the EmGFP protein. In our previous study, we have demonstrated that the expression of Nel, a glycoprotein that exerts multiple functions in neural development, can be significantly suppressed in the developing chick retina by using this technique. Our results indicate that this methodology induces a stable and robust suppression of gene expression and thus provides an efficient loss-of-function approach for studies of retinal development.
The vertebrate retina is an important model system for studying neural development. Despite its peripheral location, the retina is anatomically and developmentally an extension of the central nervous system, and the optic nerve, which consists of axons of retinal ganglion cells, represents a tract within the central nervous system. The chick retina has significant advantages as a model system to study the molecular mechanism of neural development: It is large and develops rapidly; it has structural and functional similarities to the human retina; it is highly accessible for visualization and experimental manipulations. Molecular mechanisms of cell proliferation and di....
1. Construction of miRNA expression vectors
NOTE: The procedures for constructing miRNA expression vectors (steps 1.1-1.3, 1.5-1.6.) are optimized for the miRNA expression kit, Block-iT Pol II miR RNA expression kit with EmGFP, as previously described15,16. The kit provides the expression vector designed to allow miRNA expression (pcDNA6.2-GW/EmGFP-miRNA), a control vector (pcDNA6.2-GW/EmGFP-miRNA-negative control plasmi.......
Construction of Tol2 transposon constructs for expression of artificial miRNAs against Nel
Nel (Neural Epidermal growth factor (EGF)-Like; also known as Nell2) is an extracellular glycoprotein. It has structural similarities with thrombospondin-1 and is predominantly expressed in the nervous system20,21. We have previously demonstrated that Nel regulates differentiation and survival of retinal ganglion cells
This protocol provides a detailed guide to gene silencing in the developing chick retina by transgenic expression of artificial miRNAs using in ovo electroporation and the Tol2 transposon system.
The following factors are of critical importance in performing this technique successfully. First, it is critical to use miRNA sequences that are confirmed to exert robust knockdown effects. Before applying them for in ovo electroporation, test individual pre-miRNA sequences for gene.......
The pT2K-CAGGS and pCAGGS-T2TP vectors were kindly provided by Yoshiko Takahashi (Kyoto University, Kyoto, Japan) and Koichi Kawakami (National Institute of Genetics, Mishima, Japan), respectively. We thank Michael Berberoglu for his crucial reading of the manuscript. This work was supported by grants from the Royal Society and Biotechnology and Biological Sciences Research Council (BBSRC) (UK) to M.N.....
|18 G needle, 2"
|AP-TAG kit A and AP-TAG kit B
|Q201 and Q202
|Plasmid vectors for making AP fusion proteins (https://www.genhunter.com/products/ap-tag-kit-a.html, https://www.genhunter.com/products/ap-tag-kit-b.html)
|Block-iT RNAi Designer
|An online tool to choose target sequences and design pre-miRNA sequences (https://rnaidesigner.thermofisher.com/rnaiexpress/)
|BSA 10 mg
|Capillary tubes with omega dot fiber (Micropipette needles)
|1 mm O.D. 0.75 mm I.D
|CUY21 square wave electroporator
|Diethanolamine (pH 9.8)
|Electrocompetent E. coli cells
|Fast green FCF
|Fertilized chicken eggs (Gallus gallus)
|Obtained from commercial vendors (e.g. Charles River) or local farmers
|Gooseneck fiber light source
|FuGene 6 transfection reagent
|Hamilton syringe (50 μL)
|Hamilton Cat No 80901
|Hanks' balanced salt solution
|Heavy mineral oil
|Tol2 transposase expression plasmid. A generous kind gift of Koichi Kawakami (National Institute of Genetics, Japan). Also available from Addgene.
|Pressure microinjection system
|Plasmid maxi kit
|Plasmid maxiprep kit
|Tol2 transposon vector. Kindly provided by Yoshiko Takahashi (Kyoto University, Japan)
|T4 DNA ligase
|The BLOCK-iT Pol II miR RNA expression kit with EmGFP
|Contains the miRNA expression vector (pcDNA6.2-GW/EmGFP-miRNA), a control vector (pcDNA6.2-GW/EmGFP-miRNA-negative control plasmid), accessory reagents, and instructions (https://www.thermofisher.com/order/catalog/product/K493600?SID.srch-hj-K4936-00)
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