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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Neuroscience

Loss-of-Function Approach in the Embryonic Chick Retina by Using Tol2 Transposon-Mediated Transgenic Expression of Artificial microRNAs

Published: May 18th, 2022

DOI:

10.3791/62399

1Department of Biology, Valparaiso University

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....

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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.......

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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

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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.......

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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.

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Name Company Catalog Number Comments
18 G needle, 2" VWR 89219-320
AP-TAG kit A and AP-TAG kit B GenHunter Corp 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 Invitrogen An online tool to choose target sequences and design pre-miRNA sequences (https://rnaidesigner.thermofisher.com/rnaiexpress/)
BSA 10 mg Sigma-Aldrich A2153
C115CB cables Sonidel C115CB https://www.sonidel.com/product_info.php?products_id¼254
C117 cables Sonidel C117 https://www.sonidel.com/product_info.php?products_id¼252
Capillary tubes with omega dot fiber (Micropipette needles) FHC 30-30-1 1 mm O.D. 0.75 mm I.D
CUY21 square wave electroporator Nepa Gene CUY21
Diethanolamine (pH 9.8) Sigma-Aldrich D8885
Dissecting microscope
Egg incubator Kurl B-Lab-600-110 https://www.flemingoutdoors.com/kuhl%2D%2D-600-egglaboratory-incubator%2D%2D-b-lab-600-110.html
Electrode holder Sonidel CUY580 https://www.sonidel.com/product_info.php?products_id¼85
Electrodes Nepa Gene CUY611P3-1 https://www.sonidel.com/product_info.php?products_id¼94
Electromax DH10B Invitrogen 18290-015 Electrocompetent E. coli cells
Fast green FCF Sigma-Aldrich F7258
Fertilized chicken eggs (Gallus gallus) Obtained from commercial vendors (e.g. Charles River) or local farmers
Gooseneck fiber light source
FuGene 6 transfection reagent Promega E2691
Hamilton syringe (50 μL) Sigma-Aldrich 20715 Hamilton Cat No  80901
Hanks' balanced salt solution Sigma-Aldrich H6648
Heavy mineral oil Sigma-Aldrich 330760
HEPES GIBCO 15630080
L-Homoarginine Sigma-Aldrich H10007
MgCl2 Sigma-Aldrich 13112
Micromanipulator Narishige (Japan) MM3 http://products.narishige-group.com/group1/MM-3/electro/english.html
Micropipette puller Shutter Instrument P97
p-Nitrophenylphosphate Sigma-Aldrich 20-106
PBS Sigma-Aldrich D8662
pCAGGS-T2TP vector Tol2 transposase expression plasmid. A generous kind gift of Koichi Kawakami (National Institute of Genetics, Japan). Also available from Addgene.
Pfu ThermoFisher F566S
Picospritzer (Optional) Parker Pressure microinjection system
Plasmid maxi kit Qiagen 12163 Plasmid maxiprep kit
pT2K-CAGGS vector Tol2 transposon vector. Kindly provided by Yoshiko Takahashi (Kyoto University, Japan)
PVC tubing VWR (UK) 228-3830
Spectinomycin Sigma-Aldrich S9007-5
T4 DNA ligase Promega M1801
The BLOCK-iT Pol II miR RNA expression kit with EmGFP Invitrogen K493600 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)
Thermal cycler

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