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

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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 described^15,16. 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.......

Discussion

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.......

Materials

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