Use of chick neural tube for optimizing the PSM and epithelial somites electroporation parameters: A detailed protocol
- Diethyl Pyrocarbonate (DEPC) (Sigma, cat. # 40718)
- EtOH (Sigma, cat. # 459836)
- Fast Green FCF (Sigma, cat. # F7252)
- Indian ink (Pelikan, cat. # 221143)
- Morpholino standard control oligo (5’-CCTCTTACCTCAGTTACAATTTATA-3’), Gene-Tools
- Paraformaldehyde (Sigma, cat. # 158127)
- PBS tablets (Sigma, cat. # P4417)
- pCMV-IRES-GFP (Addgene, plasmid # 78264)
- pCMV-IRES-RFP (Addgene, plasmid # 33337)
- Penicillin/Streptomycin 10000 units (Sigma, cat. # P4333)
- Qiagen EndoFree Plasmid Maxi kit (Qiagen, cat. # 12362)
- In situ hybridization (ISH) reagents: The detailed ISH protocol including reagents is publicly available from Geisha chicken embryo gene expression database .
- Chicken egg incubator with rotating shelves (Nanchang Panche Technology Co. Ltd., Model # BZ-176)
- BOD incubator (Saini Science Industries, model: SSI series)
- Stereo binocular microscope on post stand (World Precision Instruments, cat. # Z-LITE-186)
- High intensity fibre optic gooseneck illumination source (World
- Precision Instruments, cat. # PZMIII)
- Fluorescence stereomicroscope (Leica M205 FCA) equipped with AlexaFluor 488 ηm and 565 ηm filters
- Micro-torch, Piezo Butane gas burner (Gunz Dental, model GB-2001, cat. # KAM2001)
- Right-handed micromanipulator (Harvard Apparatus, cat. # EC164-0056)
- Left-handed micromanipulator (Harvard Apparatus, cat. # EC 1 60-0570)
- Mounting bases (Harvard Apparatus, cat. # EC 1 69-0225)
- Microinjector with microcapillary holder (Eppendorf FemtoJet 4i, cat. # 5252000013)
- Intracel TSS20 Ovodyne electroporator (Abbotsbury Engineering Ltd, cat. # 01-916-02)
- Intracel EP21 current amplifier (Abbotsbury Engineering Ltd, cat. # 01-918-02)
- Micropipette puller, Flaming/Brown (Harvard Apparatus, model P97)
- Electrode holder (World Precision Instruments, model M3301EH)
- NanoDrop 2000/2000c spectrophotometer (Thermo Fisher, cat. # ND-2000)
- Tubes rocker (Thermo Scientific, cat. # 11-676-333)
- Centrifuge MiniSpin (Eppendorf, cat. # 5452000018)
Miscellaneous dissection and injection tools
- Corning syringe filters, 0.2 μm (Sigma, cat. # CLS431212)
- Microcapillaries, Borosilicate thin wall with filament glass (Harvard Apparatus, cat. # 300035)
- Microloader tips (Eppendorf, cat. # 930001007)
- Pasteur pipettes (Sigma, cat. # Z331767)
- Platinum wire, 0.5 mm diameter (World Precision Instruments, cat. # PTP201)
- Tungsten wire, 0.125 mm diameter (World Precision Instruments, cat. # TGW0515)
- Watchmaker’s forceps, straight 11 cm, No. 5 (Pyramid Innovation Ltd., cat. # R35205-E)
- Watchmaker’s forceps, curved 12 cm, No. 7 (Pyramid Innovation Ltd., cat. # R35207-E)
1.1.Take an egg out of the incubator, and then wipe out the blunt end (air space side) with 70% EtOH, place the egg under the stereomicroscope with its blunt end facing up, and stick a small strip of tape on the eggshell blunt end.
1.2.Using a pair of small dissecting scissors, cut through the tape and the underneath eggshell to make a circle of 1–1.5 cm diameter egg window.
1.3.Add 2–3 drops of PBS/Penicillin-Streptomycin (10 μl/ml) on top of the outer vitelline membrane and then gently pierce the membrane using the Watchmaker’s forceps No. 5. PBS should infiltrate through the membrane which then swells and can be easily ruptured and removed by the forceps. The embryo should be immediately exposed after the membrane removal.
1.4.Inject Indian ink buffer using 1 ml syringe with 26-gauge needle underneath the blastoderm disc to visualize the embryo.
1.5.Carefully remove the inner vitelline membrane on top of the embryonic disc at the site where the tissue of interest to be electroporated using a very fine Tungsten needle.
2.Neural tube electroporation and optimization
2.1.At stage HH16 embryos, make a small egg window as described in step 1, remove the outer and inner vitelline membranes on top of the NT, and then manoeuvre and gently insert the microcapillary containing DNA mix (1–2 mg/ml IRES-GFP/Fast Green) into the NT lumen.
2.2.Fill in the NT with the DNA, gently take out the microcapillary, and then carefully place the electrodes to sandwich the NT. Add 2–3 drops of PBS/Penicillin-Streptomycin and electroporate the DNA. Add immediately 2–3 drops of PBS to cool down the electroporation site and then gently elevate the electrodes using the micromanipulator.
2.3.Test a range of volts, e.g., 15, 20, 25, 30 and 35 volts and a number of pulses, e.g., 3, 4, 5, and 6 pulses.
2.4.Aspirate some of the egg albumen using 10 ml syringe with 18-gauge needle until the embryo is slightly brought down. Seal the egg with tape and re-incubate until the desired embryo stage is reached. Embryos can be checked at any time during incubation.
2.5.Check GFP signals after 4 to 24 h of DNA electroporation. IRES-GFP can be detected by Alexa Fluor 488 ηm filter and IRES-RFP by Alexa Fluor 565 ηm filter. After incubation is complete, dissect out the electroporated embryos.
2.6.Assess the electroporation efficiency based on the following parameters (see also Table S1).
2.6.1.The overall embryo survival: should be at least 90%.
2.6.2.The embryo morphology: at least 90% of normal development should be achieved.
2.6.3.The intensity of the GFP signals: this can be classified into four levels as follows: (1) Absent (level 0): No signals can be detected; (2) Weak (level 1): Few or scattered GFP signals can be detected; (3) Moderate (level 2): Less than or 50% of the electroporated tissue shows strong GFP signals; and (4) Strong to very strong (level 3): Very bright GFP signal intensity can be easily detected in majority of the electroporated tissue.
2.6.4.Electroporation can be considered as successful when it results in a high survival rate, and normal morphology but associated with strong or very strong GFP fluorescence intensity.
3.Presegmented mesoderm electroporation
3.1.Manipulate stage HH12 embryos as described in step 1, and then manoeuvre the microcapillary containing DNA mix or MO to approach the PSM. Carefully insert the microcapillary fine tip into the rostral domain of the PSM. Inject the molecule to be electroporated which should spread in a rostrocaudal direction of the PSM until it is filled (Fig. 4A and 4B).
3.2.Place the electrodes gently to sandwich the PSM and then add 2–3 drops of PBS/Penicillin-Streptomycin before DNA is electroporated. Apply the optimized parameters obtained from the NT electroporation to electroporate the PSM (Fig. 4B and 4C).
3.3.Follow steps 2.4–2.6.
4.Epithelial somites electroporation
4.1.At stage HH16, manipulate the embryos as in step 1, manoeuvre the microcapillary to approach the first ES and gently insert the tip to pierce the somite epithelium until it reaches the somitocoel and then inject the DNA. Similarly, inject the rest of the ESs to be electroporated. Gently place the electrodes to sandwich the somites, add 2–3 drops of PBS and electroporate the epaxial domain (Fig. 5A-5C). If the hypaxial somite domain is to be electroporated, then similarly inject the somite but reverse the current as shown in Fig. 5D-5F.
4.2.After incubation is completed, dissect out the electroporated embryos and then assess the embryos’ survival, normal morphology and GFP signals intensity as described in step 2.6.
5.Embryo fixation, in situ hybridization and immunostaining
5.1.Using a pair of dissecting scissors, carefully remove the electroporated embryos in a Petri-dish containing DEPC-PBS. Carefully transfer the embryos using a 5 ml plastic Pasteur pipette with its tip cut off.
5.2.Remove the embryonic membranes using Watchmaker’s forceps No. 5 before capturing images.
5.3.Fix the embryos overnight at 4°C in 4% PFA/DEPC-PBS with rocking using a tube roller.
5.4.Wash embryos after fixation in DEPC-PBS then analyze the target gene(s) by either ISH protocol as reported in Geisha  and/or immunostaining as described by Abu-Elmagd et al. 2001 . Double ISH can be carried out as described by Abu-Elmagd et al. 2010 .
5.5. mRNA probe synthesis for ISH can be carried out as described in Geisha protocols .
ANTICIPATED RESULTS AND DISCUSSION
|Egg incubation||Not obtaining a good number of normal embryos to operate or a developmental delay||Fertilized chicken eggs are of low quality||
|Electrodes assembly and/or NT, PSM and ES electroporation||Electrodes burn the electroporation site||
|Micropipette pulling||Neels’s puller does not pull a very fine microcapillary||Pulling program is not properly optimized||
|Microcapillary assembly and/or DNA/morpholino preparation||Injected DNA or MO leaks out from the injected tissue||
|Step 2.6||Low survival rate after electroporation||
|Step 2.6||Electroporation produces kinky or malformed embryos||
|Step 2.6||GFP expression after electroporation is weak or moderate||
|Anticipated results (for the electroporated embryos after ISH)||Gene expression of a target gene (detected by ISH) is missing in embryos electroporated with a control IRES-GFP either in the injected or non-injected side||Cellular damage induced by unoptimized electroporation conditions||
- Buckingham M (2001) Skeletal muscle formation in vertebrates. Curr Opin Genet Dev 11: 440-448. doi: 10.1016/S0959-437X(00)00215-X. [View Article] [PubMed] [Google Scholar]
- Christ B, Scaal M (2008) Formation and differentiation of avian somite derivatives. Adv Exp Med Biol 638: 1-41. [PubMed] [Google Scholar]
- Bryson-Richardson RJ, Currie PD (2008) The genetics of vertebrate myogenesis. Nat Rev Genet 9: 632-646. doi: 10.1038/nrg2369. [View Article] [PubMed] [Google Scholar]
- Molkentin JD, Olson EN (1996) Defining the regulatory networks for muscle development. Curr Opin Genet Dev 6: 445-453. doi: 10.1016/S0959-437X(96)80066-9. [View Article] [PubMed] [Google Scholar]
- Andermatt I, Wilson N, Stoeckli ET (2014) In ovo electroporation of miRNA-based-plasmids to investigate gene function in the developing neural tube. Methods Mol Biol 1101: 353-368. doi: 10.1007/978-1-62703-721-1_17. [View Article] [PubMed] [Google Scholar]
- Hu X, Wang Z, Wu H, Jiang W, Hu R (2015) Ras ssDNA aptamer inhibits vascular smooth muscle cell proliferation and migration through MAPK and PI3K pathways. Int J Mol Med 35: 1355-1361. doi: 10.3892/ijmm.2015.2139. [View Article] [PubMed] [Google Scholar]
- Murai H, Tadokoro R, Sakai K, Takahashi Y (2015) In ovo gene manipulation of melanocytes and their adjacent keratinocytes during skin pigmentation of chicken embryos. Dev Growth Differ 57: 232-241. doi: 10.1111/dgd.12201. [View Article] [PubMed] [Google Scholar]
- Lopez-Sanchez C, Franco D, Bonet F, Garcia-Lopez V, Aranega A, et al. (2015) Negative Fgf8-Bmp2 feed-back is regulated by miR-130 during early cardiac specification. Dev Biol 406: 63-73. doi: 10.1016/j.ydbio.2015.07.007. [View Article] [PubMed] [Google Scholar]
- Rao M, Baraban JH, Rajaii F, Sockanathan S (2004) In vivo comparative study of RNAi methodologies by in ovo electroporation in the chick embryo. Dev Dyn 231: 592-600. doi: 10.1002/dvdy.20161. [View Article] [PubMed] [Google Scholar]
- Norris A, Streit A (2013) Morpholinos: studying gene function in the chick. Methods 66: 454-465. doi: 10.1016/j.ymeth.2013.10.009. [View Article] [PubMed] [Google Scholar]
- Gandhi S, Piacentino ML, Vieceli FM, Bronner ME (2017) Optimization of CRISPR/Cas9 genome editing for loss-of-function in the early chick embryo. Dev Biol 432: 86-97. doi: 10.1016/j.ydbio.2017.08.036. [View Article] [PubMed] [Google Scholar]
- Shirazi Fard S, Blixt M, Hallböök F (2015) Whole retinal explants from chicken embryos for electroporation and chemical reagent treatments. J Vis Exp PubMed Central PMCID: doi: 10.3791/53202. [View Article] [PubMed] [Google Scholar]
- Goljanek-Whysall K, Sweetman D, Abu-Elmagd M, Chapnik E, Dalmay T, et al. (2011) MicroRNA regulation of the paired-box transcription factor Pax3 confers robustness to developmental timing of myogenesis. Proc Natl Acad Sci U S A 108: 11936-11941. doi: 10.1073/pnas.1105362108. [View Article] [PubMed] [Google Scholar]
- Wang H, Bonnet A, Delfini MC, Kawakami K, Takahashi Y, et al. (2010) Stable, conditional, and muscle-fiber-specific expression of electroporated transgenes in chick limb muscle cells. Dev Dyn 240: 1223-1232. doi: 10.1002/dvdy.22498. [View Article] [PubMed] [Google Scholar]
- Croteau L, Kania A (2011) Optimisation of in ovo electroporation of the chick neural tube. J Neurosci Methods 201: 381-384. doi: 10.1016/j.jneumeth.2011.08.012. [View Article] [PubMed] [Google Scholar]
- Darnell DK, Kaur S, Stanislaw S, Davey S, Konieczka JH, et al. (2011) GEISHA: an in situ hybridization gene expression resource for the chicken embryo. Cytogenet Genome Res 117:30-35. [PubMed]
- Hamburger V, Hamilton HL (1951) A series of normal stages in the development of the chick embryo. J Morphol 88: 49-92. [PubMed]
- Abu-Elmagd M, Ishii Y, Cheung M, Rex M, Le Rouëdec D, et al. (2001) cSox3 expression and neurogenesis in the epibranchial placodes. Dev Biol 237: 258-269. doi: 10.1006/dbio.2001.0378. [View Article] [PubMed] [Google Scholar]
- Abu-Elmagd M, Robson L, Sweetman D, Hadley J, Francis-West P, et al. (2009) Wnt/Lef1 signaling acts via Pitx2 to regulate somite myogenesis. Dev Biol 337: 211-219. doi: 10.1016/j.ydbio.2009.10.023. [View Article] [PubMed] [Google Scholar]
- Tripathi V, Ishii Y, Abu-Elmagd MM, Scotting PJ (2009) The surface ectoderm of the chick embryo exhibits dynamic variation in its response to neurogenic signals. Int J Dev Biol 53: 1023-1033. doi: 10.1387/ijdb.082780vt. [View Article] [PubMed] [Google Scholar]
This work is licensed under a Creative Commons Attribution 3.0 License.