TY  - JOUR
TI  - Selective Enrichment and Identification of Cerebrospinal Fluid-Contacting Neurons In Vitro via PKD2L1 Promoter-Driven Lentiviral System
AU  - Tan, Wei 
AU  - Shangguan, Zeyu 
AU  - Jing, Shuwen 
AU  - Shi, Xuexing 
AU  - Li, Qizhe 
AU  - Li, Hao 
AU  - Zhang, Haodong 
AU  - Luo, Zhangrong 
AU  - Wang, Chunqing 
AU  - Dou, Xiaowei 
AU  - Li, Qing 
VL  - 15
IS  - 22
PY  - 2025
DA  - 2025/11/20
SP  - e5518
C1  - Bio-protocol 2025;15:e5518
DO  - 10.21769/BioProtoc.5518
UR  - https://doi.org/10.21769/BioProtoc.5518
AB  - Cerebrospinal fluid-contacting neurons (CSF-cNs) are a specialized group of multifunctional neurons located around the central canal of the spinal cord. They play critical roles in motor regulation, postural maintenance, and spinal cord injury repair. However, the molecular mechanisms underlying the multifunctionality of CSF-cNs remain poorly understood, partly due to the lack of established in vitro methods for their efficient selection and purification, which significantly hinders mechanistic investigations. In this study, we describe a standardized method using a PKD2L1 promoter-driven lentiviral system, which enables effective enrichment and identification of CSF-cNs in vitro through GFP labeling and puromycin selection. This protocol includes key steps such as construction of the PKD2L1 promoter-driven lentiviral vector, spinal cord tissue collection and digestion from neonatal mice, lentiviral infection, antibiotic selection, and immunofluorescence-based identification of CSF-cNs. Our method provides a reliable platform for obtaining high-purity CSF-cNs (>99%), which facilitates their functional and mechanistic studies for regenerative approaches in vitro.
KW  - Cerebrospinal fluid-contacting neurons (CSF-cNs)
KW  - Polycystic kidney disease 2-like 1 (PKD2L1)
KW  - Lentiviral system
KW  - Puromycin selection
KW  - In vitro isolation
KW  - Green fluorescent protein
KW  - Spinal cord injury
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False