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

Summary

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.

Abstract

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.

Introduction

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 vivo^1,2,3,4. In GCPs, the primary cilium acts as a pivotal Hh signal transduction hub⁵ that orchestrates the proliferation of the precursor cells^6,7,8. In vitro visualization of Hh signaling components on the pri....

Protocol

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. Refer to the Table of Materials for details about all materials used in this protocol.

1. Preexperiment preparation

1. Preparation of culture ....

Results

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.......

Discussion

Transfection of transgenes in primary GCP culture by electroporation method is typically associated with low cell viability and poor transfection efficiency^9,10. 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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Materials

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