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Updated: Jan 19, 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
Generation and Validation of Tissue-Specific Knockout Strains for Toxicology Research
Cherish A Taylor1, William Shawlot2, Jin Xiang Ren2
1Division of Pharmacology & Toxicology, College of Pharmacy, Institute for Cellular & Molecular Biology and Institute for Neuroscience, University of Texas at Austin, Austin, Texas.
Abstract:
Tissue-specific knockout mice are widely used throughout scientific research. A principle method for generating tissue-specific knockout mice is the Cre-loxP system. Here, we give a detailed description of the steps required to generate and validate tissue-specific knockout mice using the Cre-loxP system. The first protocol describes how to use gene targeting in mouse embryonic stem cells to generate mice with conditional alleles. Subsequent protocols describe how to recover Cre transgenic mice from cryopreserved sperm using in vitro fertilization and present a breeding strategy for obtaining tissue-specific knockouts. Finally, methods are provided for validating the knockout mice using PCR of genomic DNA, reverse-transcription PCR and quantitative reverse-transcription PCR of mRNA, and immunoblot analysis of proteins. © 2019 by John Wiley & Sons, Inc.
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