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Related Concept Videos

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Related Experiment Video

Updated: Jun 24, 2025

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord
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Targeted insertion of conditional expression cassettes into the mouse genome using the modified i-PITT.

Hiromi Miura1, Ayaka Nakamura2, Aki Kurosaki1

  • 1Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Kanagawa, Japan.

BMC Genomics
|June 5, 2024
PubMed
Summary

This study introduces a new method for creating genetically modified mice, enabling the insertion of conditional expression cassettes. This improves the reliability and efficiency of transgenic mouse generation for research.

Keywords:
Conditional expressionFLP-FRTMousePhiC31 integrasePronuclear injection-based targeted transgenesisTargeted transgenesis

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Area of Science:

  • Genetics
  • Molecular Biology
  • Transgenic Technology

Background:

  • Conventional transgenic (Tg) mouse generation via pronuclear injection often results in unreliable transgene expression due to random DNA insertion.
  • Pronuclear injection-based targeted transgenesis (PITT) and improved PITT (i-PITT) were developed to enhance transgene expression consistency by directing insertion to specific genomic loci.
  • A limitation of PITT is its inability to insert conditional expression cassettes using Cre-LoxP recombination due to interference between LoxP sites.

Purpose of the Study:

  • To overcome the limitation of i-PITT in inserting conditional expression cassettes.
  • To develop a modified i-PITT method that is compatible with Cre-LoxP site-specific recombination for conditional transgenesis.

Main Methods:

  • Modification of the i-PITT approach by simultaneously using PhiC31 integrase (PhiC31o) and FLP recombinase (FLPo) mRNAs.
  • Demonstration of the strategy by creating a conditional expression cassette at the Rosa26 locus in founder mice.

Main Results:

  • The modified i-PITT method successfully inserted a conditional expression cassette at the Rosa26 locus with an efficiency of 13.7%.
  • The inclusion of FLPo mRNA effectively prevented the insertion of vector backbones into the founder mice genome.

Conclusions:

  • Simultaneous application of PhiC31 integrase and FLP recombinase in the i-PITT approach enables the insertion of donor plasmids carrying Cre-LoxP-based conditional expression cassettes.
  • This advancement facilitates the generation of more reliable and versatile transgenic mouse models for biomedical research.