Generation of Mouse Haploid Somatic Cells by Small Molecules for Genome-wide Genetic Screening

Area of Science:

• Cell Biology
• Genetics
• Developmental Biology

Background:

• Mammalian haploid embryonic stem cells (haESCs) are valuable for genome-wide functional analysis.
• Spontaneous diploidization of haESCs during differentiation hinders their application in genetic studies.

Purpose of the Study:

• To investigate the mechanism of spontaneous diploidization in haESCs.
• To develop strategies for suppressing diploidization and generating stable haploid somatic cells.
• To establish a platform for large-scale genetic screening using haploid somatic cells.

Main Methods:

• Analysis of haESC diploidization mechanism, identifying metaphase mitotic slippage.
• Inhibition of Cyclin-dependent kinase 1 (CDK1) and Rho-associated protein kinase (ROCK) using small molecules.
• Generation of haploid somatic cells from all three germ layers, including neurons, via ROCK inhibition.
• Construction of a haploid neural cell library using piggyBac transposon-based insertional mutagenesis.
• Screening for Mn2+-mediated toxicity to identify genes involved in cellular response.

Main Results:

• Spontaneous diploidization of haESCs occurs in metaphase due to mitotic slippage.
• Inhibition of CDK1 and ROCK effectively suppresses diploidization.
• ROCK inhibition allows the generation of diverse haploid somatic cells, including terminally differentiated neurons.
• A genome-wide mutant haploid neural cell library was successfully created.
• The Park2 gene was identified as a key factor in Mn2+-mediated toxicity.

Conclusions:

• Small-molecule inhibition of CDK1 and ROCK prevents haESC diploidization, enabling the derivation of stable haploid somatic cells.
• This advancement extends the utility of haploid cell technology to somatic cell types for genetic screening.
• The findings provide insights into ploidy maintenance mechanisms and facilitate large-scale functional genomics in mammals.