TY - JOUR
T1 - Efficient and Cost Effective Electroporation Method to Study Primary Cilium-dependent Signaling Pathways in the Granule Cell Precursor
AU - Lo, Jason Cho Wai
AU - Wong, Wee Lin
AU - Hor, Catherine Hong Huan
N1 - Publisher Copyright:
© 2021 JoVE Journal of Visualized Experiments.
PY - 2021/11/30
Y1 - 2021/11/30
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85122904311&partnerID=8YFLogxK
U2 - 10.3791/63283
DO - 10.3791/63283
M3 - Journal article
C2 - 34927612
AN - SCOPUS:85122904311
SN - 1940-087X
VL - 2021
JO - Journal of Visualized Experiments
JF - Journal of Visualized Experiments
IS - 177
M1 - e63283
ER -