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 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 established^1,2,3.

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 zebrafish^9,10, 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 system^15,16. 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.

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 plasmid), accessory reagents, and instructions to produce miRNA expression vectors (see Table of Materials)¹⁷.

1. Designing single-stranded DNA oligos encoding pre-miRNAs against the target gene: Design single-stranded DNA oligos ("top strand" oligos (target pre-miRNAs) and "bottom strand" oligos (complements of top strand oligos)) using the online tool, RNAi Designer (see Table of Materials). See Figure 2 for the required features of the single-stranded oligos (Figure 2A) and examples of target sequences (Figure 2B).
   NOTE: It is recommended that 5-10 pre-miRNA sequences be generated for a given target gene and screened for knockdown activities in vitro (step 1.4).
2. Annealing of the top- and bottom-strand oligos to generate a double-stranded oligo
   1. Set up the following annealing reaction (Table 1) in a sterile 0.5 mL microcentrifuge tube.
   2. Incubate the reaction mixture at 95 °C for 4 min. Anneal the top- and bottom-strand oligos to generate a double-stranded oligo by allowing the reaction mixture to cool to room temperature (RT) for 5-10 min. Centrifuge the sample briefly (~5 s).
      NOTE: The annealed oligos can be stored at -20 °C without degradation for at least a year.
3. Cloning the double-stranded oligos into the miRNA expression vector (pcDNA6.2-GW/EmGFP-miRNA (Provided in the miRNA expression kit)): Clone individual double-stranded oligos into the linearized miRNA expression vector, according to the manufacturer's manual¹⁷.
4. Evaluation of knockdown effects
   NOTE: It is recommended that individual miRNA sequences be tested for gene suppression efficiency in vitro before they are applied in ovo. Knockdown efficiency can be tested by transfecting miRNA expression plasmids into a cell line that expresses the target gene. Alternatively, individual miRNA expression plasmids can be co-transfected into cell lines with an expression construct for the target gene. For target genes encoding proteins that are not membrane-anchored in their native state (e.g., soluble proteins), an alkaline phosphatase (AP) fusion protein can be used for monitoring the expression of the target protein. A cDNA sequence encoding the target protein can be fused in frame to human placental alkaline phosphatase in AP-tag vectors (APtag-1- APtag-5; see Table of Materials) and introduced into cells¹⁸. When expressed in culture cells (e.g., HEK293T cells), the AP-tagged target protein is secreted at high levels into culture media, and thus knockdown effects of miRNA sequences can be evaluated by measuring the decrease in AP activity in the culture media of miRNA-transfected cells (substeps 1.4.1-1.4.4).
   1. Culture HEK293T cells in a 24-well plate (8 x 10⁴ cells/well) overnight. Transiently transfect the cells with individual miRNA expression constructs with an expression plasmid of an AP-tagged target protein. Use the pcDNA6.2-GW/EmGFP-miRNA-negative control plasmid (provided in the miRNA expression kit) as a control. (If cell lines that stably express an AP-tagged target protein are used, transfect the cells only with individual miRNA expression constructs.)
      NOTE: A conventional lipofection reagent (see Table of Materials) is used for transfection.
   2. Collect the conditioned medium 48-72 h after the transfection and heat inactivate the endogenous AP activity in a 65 °C water bath for 5 min. Spinout debris in a desktop microcentrifuge at maximum speed for 5 min.
   3. Buffer the supernatant with 10 mM HEPES, pH 7.0 and pass it through a 0.45 µm filter.
   4. Take 100 µL (for measurement in a plate reader) or 500 µL (for a spectrophotometer) of the supernatant and add an equal amount of 2x AP substrate buffer (Table 2). Check the AP activity by measuring OD₄₀₅ in a plate reader or a spectrophotometer.
      NOTE: If the AP activity of the conditioned medium is too high for accurate measurement, dilute it with HBAH buffer (Hanks' balanced salt solution (HBSS), 0.5 mg/mL bovine serum albumin, 20 mM HEPES (pH 7.0)) or another buffer that contains a carrier protein. Do not use phosphate-containing buffers (e.g., PBS) because inorganic phosphate acts as a competitive inhibitor of AP.
5. Chaining of miRNA sequence
   NOTE: Knockdown effects can be enhanced by chaining different miRNAs against the same target gene or repeating the same miRNA. The miRNA expression vector supports the chaining of multiple pre-miRNA sequences and their co-cistronic expression¹⁷.
   1. Chain two different pre-miRNA sequences (against the same target gene) that show highest knockdown activities in in vitro assays (step 1.4), according to the manufacturer's instructions¹⁷.
   2. Evaluate gene suppression efficiency of the chained constructs using in vitro assays as described in step 1.4. If three or more pre-miRNA sequences show similarly high knockdown activities, test different combinations of two sequences and use the combinations that show the highest knockdown activity (see Representative Results).
6. Transferring the EmGFP-pre-miRNA expression cassette to the Tol2 transposon vector
   NOTE: The expression cassette containing EmGFP cDNA and two pre-miRNA sequences is transferred into the Tol2 transposon vector (pT2K-CAGGS vector, see Table of Materials). To this end, the expression cassette (encompassing from the 3' end of the CMV promoter to the miRNA reverse sequencing primer site of the miRNA expression vector) is PCR-amplified using primers with an artificial restriction enzyme site, and the PCR product is cloned into the Tol2 transposon vector. The Tol2 transposon vector contains the ubiquitous CAGGS promoter, which drives the expression of the inserted expression cassette. The CAGGS promoter and the expression cassette are flanked by the Tol2 sequences (Figure 1).
   1. PCR amplification of the EmGFP-pre-miRNA expression cassette: Follow the reaction setup and the thermocycling conditions described in Table 3.
   2. Ligate the gel-purified PCR product (c. 1.3 kb) into the restriction enzyme-digested Tol2 transposon vector. Electroporate the plasmid into competent E. coli cells (see Table of Materials) and select the recombinants (pT2K-CAGGS-EmGFP-2x miRNA constructs).
   3. Prepare the plasmid by using a conventional maxiprep kit (see Table of Materials). Check the structure and sequence of the plasmid by restriction mapping and by using the primers used for the PCR, respectively.

2. Egg storage and incubation

1. Purchase fertilized White Leghorn (Gallus gallus) eggs from local farms or commercial vendors.
   NOTE: Eggs may be kept at 12-16 °C or at 4 °C for up to 1 week prior to incubation without significant loss of viability or delay in development during incubation.
2. Label the eggs with the start date of incubation and mark the top side of the egg (where the embryo will be positioned). Incubate fertilized eggs in a horizontal position at 38 °C until the embryos have reached Hamburger and Hamilton (HH) stages 10 (33-38 h) -11 (40-45 h)¹⁹.

3. In ovo electroporation

1. Preparation for in ovo electroporation
   1. Preparation of 0.25% fast green solution: Dissolve 25 mg of Fast Green FCF in 10 mL of PBS. Filter the solution using a 0.2 µm syringe filter. The solution can be stored at RT.
   2. DNA cocktail: Prepare the injection solution by mixing the individual pT2K-CAGGS-EmGFP-2x miRNA plasmids (substep 1.6.3) with the Tol2 transposase expression plasmid (pCAGGS-T2TP vector; see Table of Materials) (5 µg/µL each) at the ratio of 2:1. Add 1/10 volume of 0.25% fast green solution to visualize the injected area.
      NOTE: The optimum DNA concentration may vary depending on the constructs.
   3. Setting up the microinjection apparatus (Figure 3A,B): The microinjection apparatus consists of the followings (see Table of Materials): Hamilton syringe, 18 G needle (Needle length = 2"), PVC (Polyvinyl chloride) tubing (2 cm length), Micropipette needle (Can be made by pulling capillary tubes with omega dot fiber (1 mm O.D. X 0.75 mm I.D) with a micropipette puller).
      1. Fill a Hamilton syringe with heavy mineral oil. Attach an 18 G needle to the syringe and fill the inner space of the needle with oil by depressing the syringe plunger.
      2. Attach a piece of 2 cm long PVC tubing to the end of the needle and fill the tubing with the oil.
      3. Attach a pulled micropipette needle to the tubing. Break off the tip of the micropipette needle to a 10-20 µm diameter by fine forceps to make a small opening. Fill the entire needle with oil.
         NOTE: Care should be taken not to trap any air bubbles in the system, as they would inhibit the flow of the DNA solution.
   4. Put 5 µL of the colored DNA cocktail (substep 3.1.2) onto a sterile Petri dish. Under the dissecting microscope, place the tip of the micropipette needle into the DNA solution on the sterile Petri dish and slowly draw the solution into the needle.
   5. Wait until the pressure equilibrates inside and outside the needle (to avoid that air goes into the needle) and take the tip of the needle out of the DNA solution. Keep the tip of the needle submerged in sterile PBS in a small beaker until injection.
   6. Setting up the electroporation apparatus (Figure 3A,C): 
      1. Set a pair of platinum electrodes with an electrode holder on a micromanipulator. Adjust the spacing between the tip of the electrodes to 2 mm (Figure 3C,E).
      2. Connect the electrodes to a square wave pulse generator with cables (see Table of Materials).
2. Microinjection of DNA solution (Figure 3D)
   1. Remove a chicken egg from the incubator and wipe the surface of the egg with a tissue paper soaked in 70% ethanol.
   2. Attach an 18 G needle to a 10 mL syringe. Insert the needle through the blunt end of the egg, angled downward (45°) to avoid damaging the yolk.
   3. Withdraw 2-3 mL of albumin from the egg. Seal the hole with a piece of Scotch tape. Confirm that the embryo and vitelline membrane are detached from the shell by "candling" the egg with light.
   4. Remove a piece of eggshell (2-3 cm diameter circle) from the top of the egg using scissors and forceps to expose the embryo. Do not window more than five eggs at any one time to prevent drying out of embryos during electroporation.
      NOTE: If it is difficult to open a window without cracking the egg, the entire top of the egg can be covered with Sellotape or Scotch tape before removing a piece of eggshell.
   5. Insert the tip of the needle into the optic vesicle from its proximal side at a 45° angle and inject the DNA cocktail by slowly depressing (or tapping on) the plunger until the blue-colored solution fills the lumen (Figure 3D).
      NOTE: Alternatively, a pressure microinjection system (see Table of Materials) can be used for the injection of DNA solutions.
   6. Withdraw the needle and place its tip back into PBS.
3. Electroporation (Figure 3E)
   1. Set pulse parameters of the electroporator as follows: Voltage: 15 V, Pulse length: 50 ms, Pulse interval: 950 ms, Pulse number: 5
   2. Add a few drops of HBSS onto the vitelline membrane over the embryo. Lower the electrodes into the HBSS using the micromanipulator, perpendicular to the anterior-posterior axis of the embryo.
      NOTE: Alternatively, this procedure can be done without adding HBSS, as albumin is a good electric conductor that allows efficient electroporation.
   3. Place the electrodes on either side (anterior (nasal) side and posterior (temporal) side) of the optic vesicle (Figure 3E). Ensure that the electrodes do not touch the embryo or blood vessels. Apply pulsed electric fields.
   4. Remove the electrodes and gently clean the electrodes with a water-soaked sterile cotton bud to avoid the accumulation of albumin.
   5. Seal the window with Scotch tape and re-incubate the embryo until the desired developmental stage.

Results

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 system^20,21. We have previously demonstrated that Nel regulates differentiation and survival of retinal ganglion cells

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

List of materials used in this article

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