Name: efficient and cost effective electroporation method to study primary cilium-dependent signaling pathways in the granule cell precursor
Uploaded: 2021-11-30T14:45:00
Description: Watch this Scientific Journal Video about Efficient and Cost Effective Electroporation Method to Study Primary Cilium-Dependent Signaling Pathways in the Granule Cell Precursor at JoVE.com

This study was supported by HKBU Seed Fund and Tier-2 Start-up Grant (RG-SGT2/18-19/SCI/009), Research Grant Council-Collaborative Research Fund (CRF-C2103-20GF) to C.H.H. Hor.

All animal-related procedures were carried out in compliance with animal handling guidelines and the protocol approved by the Department of Health, Hong Kong. Animal experiment licenses following Animal (Control of Experiments) Ordinance (Cap. 340) were obtained from the Department of Health, Hong Kong Government. The animal work was carried out in compliance with the animal safety ethics approved by HKBU Research Office and Laboratory Safety Committee.&#160;Refer to the Table of Materials for details ab...

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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+cilia+and+mammalian+Hedgehog+signaling.">Primary cilia and mammalian Hedgehog signaling.</a> Cold Spring Harbor Perspectives in Biology. 9 (5), 028175 (2017).</li><li>Hor, C. H. H., Lo, J. C. W., Cham, A. L. S., Leung, W. Y., Goh, E. L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multifaceted+functions+of+Rab23+on+primary+cilium-+and+Hedgehog+signaling-mediated+cerebellar+granule+cell+proliferation.">Multifaceted functions of Rab23 on primary cilium- and Hedgehog signaling-mediated cerebellar granule cell proliferation.</a> Journal of Neuroscience. 41 (32), 6850-6863 (2021).</li><li>Han, Y. 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Transfection of transgenes in primary GCP culture by electroporation method is typically associated with low cell viability and poor transfection efficiency9,10. This paper introduces a cost-effective and reproducible electroporation protocol that has demonstrated high efficiency and viability. In addition, we also demonstrate a straightforward method of studying the primary cilium-dependent Hh signaling pathway in primary GCP cells.
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Cerebellar GCPs are widely used to study the machinery of the Hh signaling pathway in neuronal progenitor cell-types owing to their high abundance and high sensitivity to the Hh signaling pathway in vivo1,2,3,4. In GCPs, the primary cilium acts as a pivotal Hh signal transduction hub5 that orchestrates the proliferation of the precursor cells6,7,8. In vitro visualization of Hh signaling components on the primary cilium is often challenging due to their low endogenous basal levels. Hence, transgene modification of protein expression levels and fluorophore tagging of the gene of interest are useful approaches to study the pathway at molecular resolution. However, genetic manipulation of GCP primary cultures using liposome-based transfection approaches often result in low transfection efficiency, hindering further molecular investigations9. Electroporation increases the efficiency but commonly requires exorbitant vendor-specific and cell type-restricted electroporation reagents10.
This paper introduces a high-efficiency and cost-effective electroporation method to manipulate the Hh signaling pathway components in GCP primary cultures. Using this modified electroporation protocol, a green fluorescent protein (GFP)-tagged Smoothened transgene (pEGFP-Smo) was efficiently delivered to GCPs and achieved high cell survival and transfection rates (80-90%). Furthermore, as evidenced by the immunocytochemical staining, the transfected GCPs showed high sensitivity to Smoothened agonist-induced activation of the Hh signaling pathway by trafficking EGFP-Smo to the primary cilia. This protocol shall be directly applicable and beneficial for experiments that involve in vitro genetic modification of cell types that are difficult to transfect, such as human and rodent primary cell cultures, as well as human induced&#160;pluripotent stem cells.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>GCP Culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Life Technologies LTD</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer, 70 &#181;m</td>
			<td>Corning</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I from bovine pancreas</td>
			<td>Roche</td>
			<td>11284932001</td>
			<td></td>
		</tr>
		<tr>
			<td>Earle&#8217;s Balanced Salt Solution</td>
			<td>Gibco, Life Technologies</td>
			<td>14155063</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS, qualified</td>
			<td>Thermo Scientific</td>
			<td>SH30028.02</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutamMAXTM-I ,100x</td>
			<td>Gibco, Life Technologies</td>
			<td>35050061</td>
			<td>L-glutamine substitute</td>
		</tr>
		<tr>
			<td>L-cysteine</td>
			<td>Sigma Aldrich</td>
			<td>C7352</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>BD Biosciences</td>
			<td>354277</td>
			<td>Basement membrane matrix</td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>Gibco, Life Technologies</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Papain,suspension</td>
			<td>Worthington Biochemical Corporation</td>
			<td>LS003126</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-lysine Hydrobromide</td>
			<td>Sigma Aldrich</td>
			<td>P6407</td>
			<td></td>
		</tr>
		<tr>
			<td>SAG</td>
			<td>Cayman Chemical</td>
			<td>11914-1</td>
			<td>Smoothened agonist</td>
		</tr>
		<tr>
			<td>IF staining</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma Aldrich</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary antibody mix</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GFP-goat ab</td>
			<td>Rockland</td>
			<td>600-101-215</td>
			<td>Dilution Factor: 1 : 1000</td>
		</tr>
		<tr>
			<td>Anti-Arl13b mouse monoclonal ab</td>
			<td>NeuroMab</td>
			<td>75-287</td>
			<td>Dilution Factor: 1 : 1000</td>
		</tr>
		<tr>
			<td>Anti-Pax6 rabbit polyclonal ab</td>
			<td>Covance</td>
			<td>PRB-278P</td>
			<td>Dilution Factor: 1 : 1000</td>
		</tr>
		<tr>
			<td>Secondary antibody mix</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 donkey anti-goat IgG</td>
			<td>Invitrogen</td>
			<td>A-11055</td>
			<td>Dilution Factor: 1 : 1000</td>
		</tr>
		<tr>
			<td>Alexa Fluor 555 donkey anti-mouse IgG</td>
			<td>Invitrogen</td>
			<td>A-31570</td>
			<td>Dilution Factor: 1 : 1000</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 donkey anti-rabbit IgG</td>
			<td>Invitrogen</td>
			<td>A-31573</td>
			<td>Dilution Factor: 1 : 1000</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermo Scientific</td>
			<td>62247</td>
			<td>Dilution Factor: 1 : 1000</td>
		</tr>
		<tr>
			<td>Electroporation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CU 500 cuvette chamber</td>
			<td>Nepagene</td>
			<td>CU500</td>
			<td></td>
		</tr>
		<tr>
			<td>EPA Electroporation cuvette (2 mm gap)</td>
			<td>Nepagene</td>
			<td>EC-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Life Technologies LTD</td>
			<td>31985070</td>
			<td>reduced-serum medium for transfection</td>
		</tr>
		<tr>
			<td>pEGFP-mSmo</td>
			<td>Addgene</td>
			<td>25395</td>
			<td></td>
		</tr>
		<tr>
			<td>Super Electroporator NEPA21 TYPE II In Vitro and In Vivo Electroporation</td>
			<td>Nepagene</td>
			<td>NEPA21</td>
			<td>electroporator</td>
		</tr>
	</tbody>
</table>

efficient and cost effective electroporation method to study primary cilium-dependent signaling pathways in the granule cell precursor

The primary cilium is a critical signaling organelle found on nearly every cell that transduces Hedgehog (Hh) signaling stimuli from the cell surface. In the granule cell precursor (GCP), the primary cilium acts as a pivotal signaling center that orchestrates precursor cell proliferation by modulating the Hh signaling pathway. The investigation of primary cilium-dependent Hh signaling machinery is facilitated by in vitro genetic manipulation of the pathway components to visualize their dynamic localization to the primary cilium. However, transfection of transgenes in the primary cultures of GCPs using the currently known electroporation methods is generally costly and often results in low cell viability and undesirable transfection efficiency. This paper introduces an efficient, cost-effective, and simple electroporation protocol that demonstrates a high transfection efficiency of ~80-90% and optimal cell viability. This is a simple, reproducible, and efficient genetic modification method that is applicable to the study of the primary cilium-dependent Hedgehog signaling pathway in primary GCP cultures.

Using the Opti-MEM (see the Table of Materials) as the universal reagent, this proposed electroporation methodology could achieve consistently high electroporation efficiency at ~80-90% (Figure 1). The electroporation efficiency of the Smo-EGFP vector was determined at DIV 2 post electroporation by quantification of the percentage of green fluorescence-positive cells in all paired box protein-6 (Pax6)-expressing GCP cells. The electroporation efficiency of DMSO- and SAG-trea...

Watch this Scientific Journal Video about Efficient and Cost Effective Electroporation Method to Study Primary Cilium-Dependent Signaling Pathways in the Granule Cell Precursor at JoVE.com

Efficient and Cost Effective Electroporation Method to Study Primary Cilium-Dependent Signaling Pathways in the Granule Cell Precursor

Here, we present a reproducible in vitro electroporation protocol for genetic manipulation of primary cerebellar granule cell precursors (GCPs) that is cost-effective, efficient, and viable. Moreover, this protocol also demonstrates a straightforward method for the molecular study of primary cilium-dependent Hedgehog signaling pathways in primary GCP cells.

The primary cilium is a critical signaling organelle found on nearly every cell that transduces Hedgehog (Hh) signaling stimuli from the cell surface. In ...

This protocol demonstrates a straightforward in vitro genetic modification method for the molecular study of the primary cilium-dependent signaling pathway in the primary granule cell precursor cultures. Due to the cost, low viability, and poor efficiency of the current transfection method, we introduce a simple, cost effective, and efficient electroporation technique for investigating cilium-dependent signaling pathway in primary GCP cultures. To begin, add 0.5 milliliters of culture medium into each well of the 24-well culture plate containing coated cover slips, and keep it warm at 37 degrees Celsius in a carbon dioxide incubator.Pipette the required number of cells into a sterile 1.5-milliliter microcentrifuge tube and spin at 200x G for five minutes at room temperature. Discard the supernatant and resuspend the cell pellet in 200 microliters of Opti-MEM. Repeat this procedure twice to ensure no residual culture medium is present in the tube.Set the parameters of electroporation as listed. Pipette the electroporation reaction gently to mix well, and use a long P200 pipette tip to transfer an exact volume of 100 microliters of the mixture into the two-millimeter gap cuvette. Once the cuvette is inside the cuvette chamber, press the omega button of the electroporator and note the impedance value by adhering to a precise volume of 100 microliters.The range of impedance value should be approximately 30 and 35. Press the start button to initiate the pulse. Record the measured current values in Joules shown on the reading frame.Remove the cuvette from the chamber. Immediately add 100 microliters of pre-warmed culture medium into the cuvette and resuspended it by pipetting up and down two to three times. Immediately transfer the cell suspension to the 24-well plate previously prepared.Incubate the cells at 37 degrees Celsius in a carbon dioxide incubator. Observe the cells under the fluorescence microscope on the following day. In the present study, the electroporation efficiency of DMSO and SAG-treated groups appeared comparable.Immunostaining of the primary cilium marker Arl13b demonstrates that the ciliation rate of GCP at div-2 of culture in both the vehicle and SAG-treated groups. The ciliation rate illustrates the percentage of Pax6 expressing GCPs bearing a primary cilium on the cell surface at div-2 post-electroporation. The figure shows a significant increase in smoothened EGFP localization on the primary cilium axoneme of Pax6 expression GCP cells at 24 hours post-SAG treatment, indicating a profound activation of the primary cilium-dependent hedgehog signaling pathway.In order to achieve a higher electroporation efficiency, one should ensure high purity of the plasmid used, and no residue culture media is present in the plasmid electroporation mixture. This protocol should be directly applicable and beneficial for in vitro genetic modification experiments on primary cultures and cell types that are difficult to transfect, including neurons and human-induced pluripotent stem cells.

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