Related Experiment Video
Updated: May 13, 2026
Generation of Brown Fat-Specific Knockout Mice Using a Combined Cre-LoxP, CRISPR-Cas9, and Adeno-Associated Virus Single-Guide RNA System
Published on: March 24, 2023
A non-invasive method for light-inducible knockout across all cell types in mouse subcutaneous adipose tissue
Erica de Sousa1, Magdalena Blaszkiewicz1, Kristy Townsend1
1Department of Neurological Surgery, The Ohio State University College of Medicine and Wexner Medical Center, Columbus, OH, USA.
Abstract:
Cre recombination is a widely used technique for mechanistic insights in physiology and disease. However, available constitutive and inducible Cre systems present challenges that can be prohibitive for some study designs. For example, Cre expression can result in cell types targeted across numerous tissues and organs, or when a gene is expressed across multiple cell types in a tissue, Cre-Lox restricted knockout will not enable ablation across an entire tissue or organ. Photoactivatable Cre (PA-Cre) systems enable temporally and spatially restricted gene expression control in delimited anatomical regions, typically requiring a micro-LED or fibre optic implantation. Here, we report as proof-of-concept the effective knockout of BDNF in subcutaneous adipose tissue after PA-Cre activation through external blue light illumination in awake, freely moving mice. We demonstrated that for mice with black fur, shaving can be used to anatomically limit PA-Cre activation. BDNF protein expression was decreased by 87% in the inguinal scWAT after blue light exposure, with no effect observed in the perigonadal (deep) or axillary subcutaneous (non-shaved) adipose tissues. We propose blue light induction of PA-Cre as safe and effective to study adipose tissue physiology and pathology across models. Considerations for applying this tool to future studies are also presented.
Insights
This study demonstrates blue light activation of photoactivatable Cre (PA-Cre) for targeted gene knockout in mouse adipose tissue. This method effectively reduced BDNF expression in specific subcutaneous fat depots, offering a precise tool for physiological studies.
Area of Science:
- Molecular Biology
- Genetics
- Physiology
Background:
- Cre-Lox technology is vital for studying gene function in physiology and disease.
- Existing Cre systems have limitations in spatial and temporal control, hindering specific tissue or cell-type ablation.
- Photoactivatable Cre (PA-Cre) offers advanced control but typically requires invasive light delivery methods.
Purpose of the Study:
- To establish and validate external blue light activation of PA-Cre for non-invasive, spatially restricted gene knockout.
- To demonstrate the efficacy of this method in targeting specific adipose tissue depots.
- To assess the impact on BDNF protein expression in subcutaneous adipose tissue.
Main Methods:
- Utilized PA-Cre mice and external blue light illumination for activation.
- Employed shaving to anatomically limit light exposure and PA-Cre activation in black-furred mice.
- Quantified BDNF protein levels in various adipose tissue depots post-activation using Western blotting or ELISA.
Main Results:
- Successfully achieved targeted knockout of BDNF in inguinal subcutaneous adipose tissue (scWAT) via blue light-induced PA-Cre activation.
- Demonstrated an 87% decrease in BDNF protein expression in the targeted inguinal scWAT.
- Confirmed no significant effect on BDNF levels in non-targeted perigonadal or axillary adipose tissues, highlighting spatial specificity.
Conclusions:
- External blue light induction of PA-Cre provides a safe, effective, and non-invasive method for spatially controlled gene ablation in specific adipose tissue depots.
- This technique overcomes limitations of traditional Cre systems, enabling precise investigation of adipose tissue physiology and pathology.
- The study presents a valuable proof-of-concept for future research utilizing light-inducible genetic tools in awake, freely moving animals.
