Mouse TU tagging: a chemical/genetic intersectional method for purifying cell type-specific nascent RNA

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Transcriptional profiling is crucial for studying development and disease.
• Existing cell type-specific RNA purification methods face limitations like cell dissociation trauma and incomplete RNA species identification.

Purpose of the Study:

• To introduce a novel in vivo method for covalent labeling and purification of cell type-specific RNA.
• To overcome limitations of current RNA purification techniques.

Main Methods:

• Developed "mouse thiouracil (TU) tagging," a genetic and chemical intersectional method.
• Utilized Cre-induced uracil phosphoribosyltransferase (UPRT) expression for spatial specificity.
• Employed 4-thiouracil (4TU) injection for temporal specificity, leading to thio-RNA production in UPRT(+) cells for subsequent RNA sequencing (RNA-seq).

Main Results:

• Successfully purified transcripts from spatially complex and rare cells (e.g., Tie2:Cre(+) brain endothelia/microglia).
• Captured temporally dynamic transcripts, including those induced by lipopolysaccharide (LPS).
• Differentiated immune versus niche spleen RNA using chimeric mice from UPRT(+) bone marrow transplants.

Conclusions:

• TU tagging offers a novel approach for identifying actively transcribed genes in specific cell populations.
• The method allows for precise temporal control in studying gene expression within intact organisms.
• TU tagging overcomes previous limitations, enabling comprehensive RNA analysis in complex biological systems.