A Split-Cre system designed to detect simultaneous expression of two genes based on SpyTag/SpyCatcher conjugation and...

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• The Split-Cre system allows spatiotemporal control of gene expression in vivo.
• Low activity of reconstituted NCre/CCre recombinase limits its use for simultaneous gene expression monitoring.

Purpose of the Study:

• To improve the activity of the Split-Cre system after Cre reconstitution.
• To develop a more efficient system for precise gene manipulation and analysis.

Main Methods:

• Created a Split-GFP-Cre system by fusing NCre and CCre with GFP subunits.
• Developed the Split-Spy-GCre system by incorporating SpyTag/SpyCatcher for enhanced dimerization.
• Utilized GFP-mediated dimerization and covalent SpyTag/SpyCatcher conjugation to boost Cre activity.

Main Results:

• The Split-GFP-Cre system achieved Cre recombinase activity comparable to full-length Cre in living cells.
• The Split-Spy-GCre system further increased Cre recombinase activity, especially at low expression levels.
• The dual-split system minimized issues of incomplete gene knockout and low labeling efficiency.

Conclusions:

• The enhanced Split-Spy-GCre system provides increased efficiency and robustness for genetic manipulation.
• This system enables more precise control over gene expression in specific cell subpopulations.
• It facilitates advanced analysis of gene and cell functions in complex tissues, such as the immune system.