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

Area of Science:

• Molecular Biology
• Genetics
• Neuroscience

Background:

• Adeno-associated viruses (AAVs) are widely used as non-integrating vectors for in vivo gene delivery.
• Precise control over cell-type-specific gene expression is crucial for studying gene function.
• Brown adipocytes play a key role in thermogenesis, making them an important target for metabolic research.

Purpose of the Study:

• To establish a reproducible workflow for generating Cre-dependent FLEX-AAV vectors.
• To detail methods for quantifying viral titer and performing localized injections for targeted gene delivery.
• To demonstrate the assessment of thermogenic capacity in genetically modified brown adipocytes.

Main Methods:

• Generation of Cre-dependent FLEX-AAV vectors.
• Viral titer quantification using quantitative PCR (qPCR).
• Stereotactic injections for localized delivery in mouse models.
• Assessment of brown adipocyte thermogenesis using Clark-type electrode respirometry.

Main Results:

• A robust and reproducible protocol for FLEX-AAV production was established.
• Accurate viral titer quantification methods were validated.
• Successful cell-type-specific transgene expression was achieved in mice.
• Thermogenic capacity of genetically modified brown adipocytes was successfully measured.

Conclusions:

• The presented workflow facilitates the generation and application of Cre-dependent AAVs for cell-specific gene manipulation in vivo.
• This protocol enables reliable assessment of gene function in specific cell populations, such as brown adipocytes.
• The methodology supports research into gene therapy and metabolic regulation.