Abstract
Hemizygous mutations introduced in haploid genomes can directly expose a phenotype, thus facilitating gene function analysis and forward genetic screening. Recently, mammalian haploid cells could be derived from mouse, rat, monkey, and human embryos and have been applied to screens of cellular mechanisms including cell signaling, pathogen host factors, and developmental pathways. Notably, haploid cell cultures have an intrinsic tendency for diploidization and, thus, require periodic cell sorting. Here, we report a method for rapid purification of haploid mouse embryonic stem cells from mixed cell populations with high viability and yield. Our method uses membranes with micrometer pores for force-free separation and facilitates enrichment of haploid cells without flow cytometry. The separation method simplifies maintaining haploid cell cultures and has further applications in establishing haploid cell lines from embryos and isolating cell cycle phases of mammalian cells.
Highlights
Is associated with self-diploidization,15 and it could be shown that accelerating mitosis can stabilize haHATX3 and Enhanced green fluorescent protein (EGFP) expressing diploid diHATX3 cells (haESC) to some degree.15–17 Recently, chemical inhibition of ROCK and CDK1 kinase activity has been reported to suppress diploidization and facilitate the establishment of differentiated cells with a haploid genome.18 Despite this progress, diploidization cannot be entirely prohibited and cell sorting remains essential for deriving and culturing haESCs
Based on the finding that haESCs are phenotypically smaller than diESCs,7,18 purification of haESCs from a cell mixture can be achieved by applying a forcefree separation using membranes with defined micrometer sized pores, which have been calibrated to allow haploid but not diploid cells to pass through
We previously determined that the cell size of haploid mouse embryonic stem cells (ESCs) is distinctly smaller and clearly separated from that of diploid cells
Summary
Is associated with self-diploidization, and it could be shown that accelerating mitosis can stabilize haESCs to some degree. Recently, chemical inhibition of ROCK and CDK1 kinase activity has been reported to suppress diploidization and facilitate the establishment of differentiated cells with a haploid genome. Despite this progress, diploidization cannot be entirely prohibited and cell sorting remains essential for deriving and culturing haESCs. Is associated with self-diploidization, and it could be shown that accelerating mitosis can stabilize haESCs to some degree.. Chemical inhibition of ROCK and CDK1 kinase activity has been reported to suppress diploidization and facilitate the establishment of differentiated cells with a haploid genome.. Chemical inhibition of ROCK and CDK1 kinase activity has been reported to suppress diploidization and facilitate the establishment of differentiated cells with a haploid genome.18 Despite this progress, diploidization cannot be entirely prohibited and cell sorting remains essential for deriving and culturing haESCs. Here, we present a new method for maintaining haESC cultures without the need for DNA staining or FACS. Based on the finding that haESCs are phenotypically smaller than diESCs, purification of haESCs from a cell mixture can be achieved by applying a forcefree separation using membranes with defined micrometer sized pores, which have been calibrated to allow haploid but not diploid cells to pass through.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
