Efficient bicistronic expression of cre in mammalian cells

Area of Science:

• Molecular Biology
• Genetics
• Developmental Biology

Background:

• Cre recombinase-mediated DNA recombination is crucial for generating mosaic mutant mice.
• Enhancing Cre expression is key to advancing this genetic engineering technique.

Purpose of the Study:

• To optimize the cre gene for enhanced expression in mammalian cells.
• To evaluate the efficiency of Cre recombinase in bicistronic messages.

Main Methods:

• Transient transfection assays were used to test modified cre gene constructs.
• The encephalomyocarditis virus internal ribosome entry site (IRES) was utilized in bicistronic messages.
• Recombination frequencies were quantified to assess Cre activity.

Main Results:

• Modifications like consensus sequence extension and N-terminal amino acid insertion did not alter Cre activity.
• Incorporating introns approximately doubled Cre-mediated recombination frequency.
• Bicistronic IRES-cre messages showed only a 2-fold reduction in recombination compared to monocistronic cre.

Conclusions:

• Optimized cre gene constructs, including introns, can enhance recombination efficiency.
• IRES-cre bicistronic systems allow for effective Cre recombinase expression without disrupting the targeted gene.
• This technology facilitates the generation of transgenic mouse models with tissue-specific gene expression.