Generation and Characterization of a TRIM21 Overexpressing Mouse Line

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• Understanding protein function necessitates specific protein removal.
• The Trim-Away system uses TRIM21 (TRIM-atom, RING finger, 3 and HECT domains and RLD protein 21), an antibody receptor and ubiquitin ligase, for rapid protein degradation.
• Endogenous TRIM21 expression is limited to specific cell types, restricting the application of the Trim-Away system.

Purpose of the Study:

• To generate a conditional mouse model for TRIM21 overexpression.
• To enable cell-specific TRIM21 expression for targeted protein reduction.
• To validate the Trim-Away method's efficacy in different tissues and cell types.

Main Methods:

• CRISPR technology was used to insert a conditional TRIM21 overexpression cassette into the Rosa26 safe harbor site.
• Generated mice were bred with Cre-driver lines (Zp3^Cre for oocytes, Hprt^Cre for global expression) to achieve cell-specific TRIM21 overexpression.
• Trim-Away system efficacy was tested by microinjecting antibodies against IP₃ receptor or SNAP23 in oocytes and electroporating antibodies in neural cells.

Main Results:

• TRIM21 protein was specifically overexpressed in oocytes using Zp3^Cre mice and globally using Hprt^Cre mice.
• Microinjection of specific antibodies into TRIM21-overexpressing oocytes resulted in effective degradation of IP₃ receptor and SNAP23.
• Cortical neural cells from globally-overexpressing TRIM21 mice showed rapid reduction of IP₃ receptor protein after antibody electroporation.

Conclusions:

• The developed conditional TRIM21-overexpressing mouse lines effectively enable the Trim-Away system for targeted protein reduction.
• This method allows for acute and specific degradation of proteins in a wide range of cell types and tissues.
• These mice represent a valuable tool for the research community to study protein function through targeted degradation.