Genotyping Protocols for Genetically Engineered Mice.

Area of Science:

• * Genetics and Genomics
• * Mammalian Model Systems
• * Molecular Biology

Background:

• * Laboratory mice are crucial for studying gene function and human disease modeling.
• * Advancements in gene editing technologies like CRISPR have enabled precise mouse genome manipulation.
• * Standardized methods are essential for consistent identification of genetically modified mouse models across research facilities.

Purpose of the Study:

• * To provide comprehensive guidelines for identifying genetically modified mouse models.
• * To detail endpoint polymerase chain reaction (PCR) methods for genotyping.
• * To outline strategies for identifying models created using newer gene-editing technologies, including CRISPR.

Main Methods:

• * Genomic DNA extraction from various mouse tissues and samples (tail biopsy, ear punch, blastocysts, semen, blood, buccal swabs).
• * Endpoint polymerase chain reaction (PCR) for routine genotyping.
• * CRISPR-specific assays including T7E1/Surveyor, off-target mutation detection, and gene knock-in/deletion analysis.

Main Results:

• * Established protocols for reliable DNA purification from diverse biological samples.
• * Demonstrated the efficacy of endpoint PCR for routine genotyping of genetically modified mice.
• * Presented methods for verifying CRISPR-mediated edits, including insertion/deletion detection and off-target analysis.

Conclusions:

• * Standardized molecular techniques are vital for accurate characterization of genetically modified mouse models.
• * Endpoint PCR remains a robust method for genotyping, complemented by specialized assays for CRISPR-edited models.
• * These guidelines facilitate consistent and reproducible research using genetically engineered mice.