Unmodified Cre recombinase crosses the membrane

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• Site-specific recombination is crucial for genetic engineering.
• Traditional methods using Cre recombinase expression vectors can cause unwanted cellular effects.
• Developing direct delivery methods for recombinases is essential for precise genetic manipulation.

Purpose of the Study:

• To investigate the cell-permeability of Cre recombinase for direct use in lox-specific recombination.
• To evaluate the intrinsic membrane-crossing ability of unmodified Cre recombinase.
• To assess the potential of Cre recombinase for in vivo gene editing applications.

Main Methods:

• Utilized a cell line engineered to report loxP-specific recombination via fluorescence change (red to green).
• Compared purified recombinant Cre recombinase with and without a protein transduction domain.
• Applied purified Cre enzyme to primary bone marrow cells from C/EBPalpha(fl/fl) mice.

Main Results:

• Unmodified Cre recombinase demonstrated intrinsic cell membrane permeability.
• Direct addition of Cre enzyme induced loxP-specific recombination in the reporter cell line.
• Excision of the floxed C/EBPalpha gene was observed in primary mouse bone marrow cells.

Conclusions:

• Cre recombinase possesses inherent cell-penetrating properties, eliminating the need for fusion domains.
• Direct application of Cre enzyme enables efficient site-specific recombination in mammalian cells.
• Cre recombinase is a promising tool for direct genetic manipulation in vitro and in vivo.