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Method Article
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 differentiation, morphogenesis, and axon guidance during neural development have been extensively studied by using the chicken retina.
In ovo electroporation has been successfully used over the last two decades to introduce ectopic genes into cells in the developing chick embryo. This technique allows for labeling of developing cells, cell fate tracing, and tracing of cell migration and axon tracts, as well as ectopic gene expression for in vivo analysis of gene function. The conditions of in ovo electroporation for efficient ectopic gene expression in chick embryos have been well established1,2,3.
Despite these advantages, the lack of a stable loss-of-function technique for studies of gene function had been a major technical limitation of the chick embryo. Whereas chick embryos electroporated with small interfering RNAs (siRNAs)4 or expression vectors for short hairpin RNAs (shRNAs)5 show knockdown of the targeted gene, gene suppression in those approaches is transient because the effects disappear once cells lose the introduced RNAs or DNAs. A more stable gene suppression can be achieved by delivering siRNAs into chick embryos by an RCAS (Replication Competent Avian sarcoma-leukosis virus (ASLV) long terminal repeat (LTR) with a Splice acceptor) retrovirus system6. The viral vector integrates into the host genome, and the ectopic genes are stably expressed. However, the retrovirus can only integrate into the genome of dividing cells during the mitotic (M) phase of the cell cycle, which may impose a limitation on the developmental stages and/or cell types for which this loss-of-function approach can be applied. In addition, expression of transgenes by RCAS appears slower and less robust than that induced by in ovo electroporation7.
Transposons are genetic elements that move from one location on the genome to another. The Tol2 element is a member of the hAT transposable element family and contains an internal gene encoding a transposase that catalyzes the transposon reaction of the Tol2 element8. When a plasmid vector that carries a gene expression cassette flanked by the sequences of the left and right ends of the Tol2 elements (200 bp and 150 bp, respectively) is introduced into vertebrate cells with a Tol2 transposase expression construct, the expression cassette is excised from the plasmid and integrated into the host genome, which supports a stable expression of the ectopic gene (Figure 1). It has been shown that the Tol2 transposable element can induce gene transposition very efficiently in different vertebrate species, including zebrafish9,10, frogs11, chicks12, and mice13, and thus is a useful method of transgenesis and insertional mutagenesis. The Tol2 transposon system has been successfully used for conditional knockdown of a target gene by genomic integration of siRNA that is processed from long double-stranded RNA14.
This protocol describes a loss-of-function approach in the chick embryo that involves the introduction of artificial microRNAs (miRNAs) by the Tol2 transposon system15,16. In this approach, an expression cassette for the EmGFP (emerald green fluorescent protein) marker and artificial miRNAs against a target gene is cloned into a Tol2 transposon vector. The Tol2 transposon construct is then 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 studies, we successfully knocked down the expression of Nel, an extracellular glycoprotein predominantly expressed in the nervous system, in the developing chick retina (see Representative Results). Our results indicate that stable and efficient gene suppression can be achieved in ovo by this technique.
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 plasmid), accessory reagents, and instructions to produce miRNA expression vectors (see Table of Materials)17.
2. Egg storage and incubation
3. In ovo electroporation
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 authors have nothing to disclose.
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.
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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