Reporter-Based Synthetic Genetic Array Analysis: A Functional Genomics Approach for Investigating Transcript or...

Area of Science:

• Yeast genetics
• Molecular biology
• High-throughput screening

Background:

• Fluorescent reporter genes are crucial for measuring cellular components like transcripts and proteins.
• Synthetic genetic array (SGA) analysis is a powerful tool for studying gene interactions in yeast.
• Existing methods require adaptation for simultaneous, large-scale fluorescent readout quantification.

Purpose of the Study:

• To introduce reporter synthetic genetic array (R-SGA) analysis, a novel method for high-throughput quantification of fluorescent protein readouts.
• To demonstrate the applicability of R-SGA across various yeast mutant collections.
• To provide a detailed protocol for implementing R-SGA and analyzing its output.

Main Methods:

• Integration of a fluorescent reporter system with standard SGA analysis in an automated pipeline.
• Application of R-SGA to screen deletion, temperature-sensitive, and inducible overexpression yeast mutant collections.
• Development of an alternative analysis pipeline using flow cytometry for liquid cultures.

Main Results:

• R-SGA successfully enables simultaneous quantification of fluorescent protein readouts across thousands of yeast strains.
• The method is adaptable to diverse array-based yeast strain collections.
• Established protocols for both array-based and flow cytometry analysis pipelines, including data normalization strategies.

Conclusions:

• R-SGA analysis offers a robust and versatile platform for large-scale genetic screening in yeast.
• This methodology significantly enhances the capacity for studying gene function and interactions through fluorescent reporters.
• R-SGA provides a valuable tool for the yeast research community, enabling deeper insights into cellular processes.