A reversible haploid mouse embryonic stem cell biobank resource for functional genomics

Area of Science:

• Genetics
• Stem Cell Biology
• Molecular Biology

Background:

• Functional genomics research is essential but often hindered by clonal variation in cell populations.
• Reproducibility in biological studies is a significant challenge due to inherent cellular heterogeneity.

Purpose of the Study:

• To establish a comprehensive resource for functional annotation of the genome.
• To develop a method overcoming clonal variance for high-throughput genetic screens.
• To identify novel genes involved in essential cellular processes, angiogenesis, and viral infections.

Main Methods:

• Genome-saturated mutagenesis was employed to generate a biobank of over 100,000 haploid mouse embryonic stem (mES) cell lines.
• Each cell line features genetically barcoded, conditional, and reversible mutations targeting 16,970 genes.
• Reversible mutagenesis was utilized to enable direct functional annotation in sister cells, mitigating clonal variance.

Main Results:

• The Haplobank, a resource of hemi/homozygous mutant mES cells, was created and made available to researchers.
• Reverse genetic screens using the Haplobank identified genes regulating essential processes in mES cells, sprouting angiogenesis, and blood vessel lineage specification.
• A genome-wide forward screen identified PLA2G16 as a host factor crucial for rhinovirus-induced cytotoxicity.

Conclusions:

• The Haplobank combined with reversible mutagenesis technology provides a powerful platform for high-throughput and reproducible functional genome annotation.
• This resource significantly advances the ability to uncover gene functions and their contributions to various biological phenotypes.
• The study highlights the utility of the Haplobank in discovering genes involved in fundamental biological processes and disease mechanisms.