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

Abstract

The ability to directly uncover the contributions of genes to a given phenotype is fundamental for biology research. However, ostensibly homogeneous cell populations exhibit large clonal variance that can confound analyses and undermine reproducibility. Here we used genome-saturated mutagenesis to create a biobank of over 100,000 individual haploid mouse embryonic stem (mES) cell lines targeting 16,970 genes with genetically barcoded, conditional and reversible mutations. This Haplobank is, to our knowledge, the largest resource of hemi/homozygous mutant mES cells to date and is available to all researchers. Reversible mutagenesis overcomes clonal variance by permitting functional annotation of the genome directly in sister cells. We use the Haplobank in reverse genetic screens to investigate the temporal resolution of essential genes in mES cells, and to identify novel genes that control sprouting angiogenesis and lineage specification of blood vessels. Furthermore, a genome-wide forward screen with Haplobank identified PLA2G16 as a host factor that is required for cytotoxicity by rhinoviruses, which cause the common cold. Therefore, clones from the Haplobank combined with the use of reversible technologies enable high-throughput, reproducible, functional annotation of the genome.

Parallel PRC2/cPRC1 and vPRC1 pathways silence lineage-specific genes and maintain self-renewal in mouse embryonic stem cells

“…First, we evaluated the role of PRC2 in transcriptional silencing and self-renewal by examining Eed-null haploid mESCs from the Haplobank repository (51). Haploid mESCs are indistinguishable from their diploid counterparts with regard to growth and developmental potential but facilitate reverse genetics via gene-trap insertion (51). Gene-trap disruption is reversible, restoring normal gene expression and allowing the exclusion of unspecific secondary mutations ( fig.…”

“…All mESCs used in this study were derived from haploid HMSc2 termed AN3-12 (51). mESCs grown under serum conditions were cultivated feeder free in base medium composed of high-glucose Dulbecco's Modified Eagle Medium supplemented with 13.5% fetal bovine se-rum (FBS; Sigma), 10 mM Hepes (pH 7.4), 2 mM GlutaMAX (Gibco), 1 mM sodium pyruvate (Sigma), penicillin (100 U/ml) (Sigma), streptomycin (0.1 mg/ml) (Sigma), 1× nonessential amino acids (Sigma), 50 mM -mercaptoethanol (Gibco) supplemented with 10% FBS (Sigma), and recombinant leukemia inhibitory factor (LIF; produced in-house).…”

“…Eed-null mESCs, generated by gene-trap insertion, were obtained from the Haplobank repository (51). Briefly, translation of full-length Eed was terminated by integration of a retrovirus-based enhanced gene-trap cassette between exons 2 and 3.…”

Cryo-EM structure of pleconaril-resistant rhinovirus-B5 complexed to the antiviral OBR-5-340 reveals unexpected binding site

“…However, due to the insufficient efficacy, mutational escape, and/or side effects observed in clinical studies, none of these antivirals has been approved (15)(16)(17). Recently, it was discovered that the small cellular phospholipase pla2g16 aids the transfer of the viral RNA genome into the cytosol (18,19). It may be a potential host target for the treatment of RVs and enteroviruses (EVs).…”

Genetic screening and multipotency in rhesus monkey haploid neural progenitor cells

“…haESCs have been practically applied to discover genes that are targeted by toxins (Elling et al, 2011) and pluripotency exit genes (Leeb et al, 2014). haESCs have even served as a mutant bio-bank for tracing unknown genes in many interesting biological processes (Elling et al, 2017). However, haESCs tend to diploidize during proliferation and differentiation, thus necessitating periodic sorting for haploids.…”