Derivation and maintenance of mouse haploid embryonic stem cells

Elling, Ulrich; Woods, Michael; Forment, Josep V.; Fu, Beiyuan; Yang, Fengtang; Ng, Bee Ling; J, Vicente; Adams, David J.; Doe, Brendan; Jackson, Stephen; Penninger, Josef; Balmus, Gabriel

doi:10.1038/s41596-019-0169-z

Cited by 17 publications

(15 citation statements)

References 53 publications

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“…The generation of a comprehensive cell bank of haploid AN3-12 cells, containing insertions in 16,970 mouse genes, was already created and described elsewhere ( Elling et al, 2019 ; https://www.haplobank.at/ecommerce/control/main ). In short, for retroviral library generation, enhanced gene-trap (EGT) viruses carrying a neomycin-resistance cassette were packaged in PlatinumE (Cell Biolabs) cells.…”

Section: Methodsmentioning

confidence: 99%

GFPT2/GFAT2 and AMDHD2 act in tandem to control the hexosamine pathway

Kroef

Ruegenberg

Horn

et al. 2022

eLife

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The hexosamine biosynthetic pathway (HBP) produces the essential metabolite UDP-GlcNAc and plays a key role in metabolism, health, and aging. The HBP is controlled by its rate-limiting enzyme glutamine fructose-6-phosphate amidotransferase (GFPT/GFAT) that is directly inhibited by UDP-GlcNAc in a feedback loop. HBP regulation by GFPT is well studied but other HBP regulators have remained obscure. Elevated UDP‑GlcNAc levels counteract the glycosylation toxin tunicamycin (TM) and thus we screened for TM resistance in haploid mouse embryonic stem cells (mESCs) using random chemical mutagenesis to determine alternative HBP regulation. We identified the N‑acetylglucosamine deacetylase AMDHD2 that catalyzes a reverse reaction in the HBP and its loss strongly elevated UDP-GlcNAc. To better understand AMDHD2, we solved the crystal structure and found that loss-of-function is caused by protein destabilization or interference with its catalytic activity. Finally, we show that mESCs express AMDHD2 together with GFPT2 instead of the more common paralog GFPT1. Compared with GFPT1, GFPT2 had a much lower sensitivity to UDP-GlcNAc inhibition, explaining how AMDHD2 loss-of-function resulted in HBP activation. This HBP configuration in which AMDHD2 serves to balance GFPT2 activity was also observed in other mESCs and, consistently, the GFPT2:GFPT1 ratio decreased with differentiation of human embryonic stem cells. Together, our data reveal a critical function of AMDHD2 in limiting UDP‑GlcNAc production in cells that use GFPT2 for metabolite entry into the HBP.

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Section: Methodsmentioning

confidence: 99%

GFPT2/GFAT2 and AMDHD2 act in tandem to control the hexosamine pathway

Kroef

Ruegenberg

Horn

et al. 2022

eLife

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“…Experimental details of genomic DNA extraction, restriction digest, ring-ligation and inverse PCR with primers located in the genetrap (see Fig. S3A and B), as well as next generation sequencing of integration sites were described previously (27). In short, the cell clones that were resistant to infection with RVFV MP-12 were trypsinized, washed in PBS and incubated overnight in gDNA Lysis buffer (GDLB: 10mM Tris-HCl pH 8.0, 5mM EDTA, 100mM NaCl, 1% SDS, freshly added 1 mg/mL Proteinase K).…”

Section: Mapping Of Genomic Genetrap Vector Integration Sitesmentioning

confidence: 99%

“…Medium and supplements were purchased from Thermo Fisher Scientific. The mouse haploid embryonic stem cells AN3-12 were derived and sorted for their haploidy as described (26,27).…”

Section: Cells and Virusesmentioning

confidence: 99%

Low Density Lipoprotein Receptor-Related Protein 1 (LRP1) as an auxiliary host factor for RNA viruses including SARS-CoV-2

Devignot

Sha

Burkard

et al. 2022

Preprint

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Viruses with an RNA genome are the main causes of zoonotic infections. In order to identify novel pro-viral host cell factors, we screened a haploid insertion-mutagenized mouse embryonic cell library for clones that rendered them resistant to the zoonotic Rift Valley fever virus (RVFV; family Phleboviridae, order Bunyavirales). This screen returned the Low Density Lipoprotein Receptor-Related protein 1 (LRP1, or CD91) as top hit, a 600 kDa plasma membrane protein known to be involved in a wide variety of cell activities. Inactivation of LRP1 expression in human cells reduced RVFV infection at the early stages of infection, including the particle attachment to the cell. In the highly LRP1-positive human HuH-7 cell line, LRP1 was required for the early infection stages also of Sandfly fever Sicilian virus (SFSV; family Phleboviridae, order Bunyavirales), vesicular stomatitis (VSV; family Rhabdoviridae, order Mononegavirales), Encephalomyocarditis virus (EMCV, family Picornaviridae), and the coronaviruses MERS-CoV, SARS-CoV-1, and SARS-CoV-2. While for RVFV, EMCV, and MERS-CoV the replication cycle could eventually catch up, LRP1 requirement for the late infection stage in HuH-7 cells was observed for SFSV, La Crosse virus (LACV; family Peribunyaviridae, order Bunyavirales), VSV, SARS-CoV-1, and SARS-CoV-2. For SARS-CoV-2, the absence of LRP1 stably reduced viral RNA levels in human lung Calu-3 cells, and both RNA levels and particle production in the hepatic HuH-7 cells. Thus, we identified LRP1 as a host factor that supports various infection cycle stages of a broad spectrum of RNA viruses.

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“…At present, scientists mainly focus on the establishment of haploid embryonic stem cells [114,115]. In 2012, Hui Y [116] and Wei Li [117] reported the derivation of androgenetic haploid ES (ahES) cells from androgenetic blastocysts, and they obtained live mice upon injection of ahES cells into MII oocytes.…”

Section: Progress Of Dscmentioning

confidence: 99%

Double sperm cloning (DSC) is a promising strategy in mammalian genetic engineering and stem cell research

Zhang

Huang

et al. 2020

Stem Cell Res Ther

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Embryonic stem cells (ESCs) derived from somatic cell nuclear transfer (SCNT) and induced pluripotent stem cells (iPSCs) are promising tools for meeting the personalized requirements of regenerative medicine. However, some obstacles need to be overcome before clinical trials can be undertaken. First, donor cells vary, and the reprogramming procedures are diverse, so standardization is a great obstacle regarding SCNT and iPSCs. Second, somatic cells derived from a patient may carry mitochondrial DNA mutations and exhibit telomere instability with aging or disease, and SCNT-ESCs and iPSCs retain the epigenetic memory or epigenetic modification errors. Third, reprogramming efficiency has remained low. Therefore, in addition to improving their success rate, other alternatives for producing ESCs should be explored. Producing androgenetic diploid embryos could be an outstanding strategy; androgenic diploid embryos are produced through double sperm cloning (DSC), in which two capacitated sperms (XY or XX, sorted by flow cytometer) are injected into a denucleated oocyte by intracytoplasmic sperm injection (ICSI) to reconstruct embryo and derive DSC-ESCs. This process could avoid some potential issues, such as mitochondrial interference, telomere shortening, and somatic epigenetic memory, all of which accompany somatic donor cells. Oocytes are naturally activated by sperm, which is unlike the artificial activation that occurs in SCNT. The procedure is simple and practical and can be easily standardized. In addition, DSC-ESCs can overcome ethical concerns and resolve immunological response matching with sperm providers. Certainly, some challenges must be faced regarding imprinted genes, epigenetics, X chromosome inactivation, and dosage compensation. In mice, DSC-ESCs have been produced and have shown excellent differentiation ability. Therefore, the many advantages of DSC make the study of this process worthwhile for regenerative medicine and animal breeding.

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Derivation and maintenance of mouse haploid embryonic stem cells

Cited by 17 publications

References 53 publications

GFPT2/GFAT2 and AMDHD2 act in tandem to control the hexosamine pathway

GFPT2/GFAT2 and AMDHD2 act in tandem to control the hexosamine pathway

Low Density Lipoprotein Receptor-Related Protein 1 (LRP1) as an auxiliary host factor for RNA viruses including SARS-CoV-2

Double sperm cloning (DSC) is a promising strategy in mammalian genetic engineering and stem cell research

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