# Protocol for stable cell line production to express muscle-type nicotinic receptor

## TL;DR

This paper provides a detailed protocol for creating stable cell lines that produce muscle-type nicotinic receptors, useful for cryo-EM studies.

## Contribution

A new doxycycline-inducible protocol for co-expressing all four subunits of muscle-type nicotinic acetylcholine receptor in stable cell lines.

## Key findings

- The protocol enables sufficient AChR production for single-particle cryo-EM.
- The method includes lentivirus infection, puromycin selection, and FACS-based isolation of clones.
- The approach may be applicable to other hetero-multimeric protein complexes.

## Abstract

The adult muscle-type nicotinic acetylcholine receptor (AChR) is essential for neuromuscular transmission but is difficult to produce due to the requirement for coordinated subunit assembly. Here, we present a protocol for generating doxycycline-inducible stable cell lines that co-express all four subunits. We describe steps for lentivirus infection, puromycin selection, fluorescence-activated cell sorting, clonal expansion, and protein expression test. This protocol enables AChR production at quantities sufficient for single-particle cryoelectron microscopy (cryo-EM) and may be applicable to other hetero-multimeric protein complexes.

For complete details on the use and execution of this protocol, please refer to Li et al.1

•Guidance on designing lentivirus constructs to express human muscle-type AChR subunits•Steps for lentiviral co-infection and antibiotic selection for stably integrated cells•Instructions for FACS isolation of clones expressing all AChR subunits•Procedures for AChR expression test by 125I-α-bungarotoxin binding assay

Guidance on designing lentivirus constructs to express human muscle-type AChR subunits

Steps for lentiviral co-infection and antibiotic selection for stably integrated cells

Instructions for FACS isolation of clones expressing all AChR subunits

Procedures for AChR expression test by 125I-α-bungarotoxin binding assay

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

The adult muscle-type nicotinic acetylcholine receptor (AChR) is essential for neuromuscular transmission but is difficult to produce due to the requirement for coordinated subunit assembly. Here, we present a protocol for generating doxycycline-inducible stable cell lines that co-express all four subunits. We describe steps for lentivirus infection, puromycin selection, fluorescence-activated cell sorting, clonal expansion, and protein expression test. This protocol enables AChR production at quantities sufficient for single-particle cryoelectron microscopy (cryo-EM) and may be applicable to other hetero-multimeric protein complexes.