# Protocol for targeted gene manipulation and thermogenic evaluation in mouse brown adipocytes

## TL;DR

This paper provides a detailed protocol for using AAVs to manipulate genes in mouse brown fat cells and measure their thermogenic activity.

## Contribution

A reproducible workflow for generating and using Cre-dependent FLEX-AAVs in mouse brown adipocytes with thermogenic evaluation.

## Key findings

- A protocol for cloning genes into FLEX cassettes and preparing high-titer AAVs in HEK293T/17 cells.
- Guidelines for injecting AAVs into interscapular brown adipose tissue in mice.
- Steps for measuring thermogenesis in modified adipocytes using respirometry.

## Abstract

Adeno-associated viruses (AAVs) are versatile, non-integrating vectors for in vivo gene delivery. We present a reproducible workflow for generating Cre-dependent FLEX-AAVs, quantifying viral titer, and performing localized injections for cell-type-specific transgene expression in mice. The protocol also details the assessment of thermogenic capacity in genetically modified brown adipocytes using Clark-type electrode respirometry.

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

•Instructions for cloning of a gene of interest into a FLEX cassette•Procedures for high-titer AAV preparation in HEK293T/17 cells•Guidelines for surgical AAV injection into interscapular brown adipose tissue•Steps for assessing thermogenesis of modified adipocytes using respirometry

Instructions for cloning of a gene of interest into a FLEX cassette

Procedures for high-titer AAV preparation in HEK293T/17 cells

Guidelines for surgical AAV injection into interscapular brown adipose tissue

Steps for assessing thermogenesis of modified adipocytes using respirometry

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

Adeno-associated viruses (AAVs) are versatile, non-integrating vectors for in vivo gene delivery. We present a reproducible workflow for generating Cre-dependent FLEX-AAVs, quantifying viral titer, and performing localized injections for cell-type-specific transgene expression in mice. The protocol also details the assessment of thermogenic capacity in genetically modified brown adipocytes using Clark-type electrode respirometry.