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Related Concept Videos

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The recognition sites for Cre recombinase called LoxP...
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Related Experiment Video

Updated: Sep 24, 2025

Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format
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One-Step Genotyping Method in loxP-Based Conditional Knockout Mice Generated by CRISPR-Cas9 Technology.

He Zhu1, Siqian Liu1, Wenxi He2

  • 1Department of Respiratory and Critical Care Medicine, The Center for Biomedical Research, NHC Key Laboratory of Respiratory Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, China.

Molecular Biotechnology
|May 3, 2022
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Summary
This summary is machine-generated.

This study introduces a rapid and reliable tetra primer-paired PCR method for genotyping genetically modified mice. This technique efficiently distinguishes wild-type, loxP, and heterozygous genotypes, improving accuracy in large-scale mouse model studies.

Keywords:
GenotypingPCR-based protocolTetra primer-paired PCR amplificationloxP allele

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Area of Science:

  • Genetics
  • Molecular Biology
  • Biotechnology

Background:

  • CRISPR-Cas9 and in vitro fertilization (IVF) enable efficient generation of genetically modified mouse models.
  • Traditional genotyping methods for loxP-based strategies are often time-consuming and lack reliability due to the small size of the loxP site.
  • Accurate and efficient genotyping is crucial for large-scale mouse model studies.

Purpose of the Study:

  • To develop an efficient and reliable genotyping method for genetically modified mice using a loxP-based strategy.
  • To simplify genotype interpretation and enhance accuracy, especially for large-scale experiments.
  • To address the limitations of existing time-consuming and unreliable genotyping techniques.

Main Methods:

  • Development of a tetra primer-paired PCR amplification technique.
  • Simultaneous amplification of internal control, wild-type (wt), and loxP genotype bands in a single PCR reaction.
  • Analysis of band patterns for straightforward genotype determination (wt/wt, wt/loxP, loxP/loxP).

Main Results:

  • The tetra primer-paired PCR method successfully generated distinct bands for internal control, wild-type, and loxP genotypes.
  • Mouse genotypes could be easily interpreted from the observed band patterns.
  • Relatively constant band ratios in different genotypes aided in excluding minor cross-contamination, enhancing reliability.

Conclusions:

  • The described tetra primer-paired PCR method offers an efficient and reliable solution for genotyping genetically modified mice.
  • This technique simplifies genotype analysis and improves accuracy, making it suitable for high-throughput applications.
  • The method enhances the reliability of genotyping, particularly in large-scale mouse model generation and validation.