Targeted insertion of conditional expression cassettes into the mouse genome using the modified i-PITT

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.