Generation of Connexin-Expressing Stable Cell Pools

Area of Science:

• Molecular Biology
• Cell Biology
• Genetics

Background:

• Stable cell pools offer reproducible transgene transmission compared to transient expression.
• Inducible transgene expression is crucial for studying gene function in various assays.
• Lentivirus (LV) facilitates transgene delivery, genomic integration, and expression in diverse cell types.

Purpose of the Study:

• To develop a lentivirus vector for generating doxycycline-inducible stable cell pools.
• To establish a protocol for studying gene of interest (GOI) function, particularly connexins like Cx32.
• To enable functional assays, including Ca2+ imaging with GCaMP6s.

Main Methods:

• Development of a doxycycline-inducible bicistronic lentivirus transfer vector.
• Production of LV particles via transient transfection and generation of stable cell pools.
• Step-by-step immunolabeling protocols for confocal microscopy of transgene expression in living and fixed cells.

Main Results:

• Successful generation of stable cell pools using the developed LV vector.
• Demonstration of Cx32 protein expression and localization via immunolabeling.
• Validation of the protocol for functional assays like Ca2+ imaging.

Conclusions:

• The developed lentivirus vector and protocol provide a robust tool for creating stable cell pools.
• This method facilitates the study of gene function using various molecular, immune, and optical assays.
• The protocol is adaptable for expressing any GOI for both in vitro and in vivo research.