SAMHD1 Knockout iPSC model enables high lenti-viral transduction in myeloid cell types
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ABSTRACT:
PROVIDER: PRJNA1224261 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR32360300.fastq.gz | Fastqsanger.gz | |||
| SRR32360301.fastq.gz | Fastqsanger.gz | |||
| SRR32360302.fastq.gz | Fastqsanger.gz | |||
| SRR32360303.fastq.gz | Fastqsanger.gz | |||
| SRR32360304.fastq.gz | Fastqsanger.gz |
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Project description:Recent advances in functional genomics tools have ushered in a new era of genetic editing to identify molecular pathways relevant to developmental and disease biology. However, limited model systems are available that adequately mimic cell states and phenotypes associated with human disease pathways. Here, we quantitatively analyzed the founder population bottleneck effect and demonstrated how the population changes from induced pluripotent stem cells (iPSCs) to hematopoietic stem cells and to the final induced macrophage population. We then engineered SAMHD1 knockout (KO) iPSC and characterized the iPSC line with RNA Seq, and induced macrophages from two distinct protocols with functional analysis. We then generated SAMHD1 KO CRISPR-dCAS9 KRAB iPSC through lenti-viral transduction aiming to increase the efficiency of lentiviral mediated gene transfer. We demonstrated increased lenti-viral transduction efficiency in induced macrophage, as well as microglia induced with two distinct protocols. This model allows for efficient gene knock down, as well as large-scale functional genomics screens in mature iPSC-derived macrophages or microglia with applications in innate immunity and chronic inflammatory disease biology. These experiments highlight the broad applicability of this platform for disease-relevant target identification and may improve our ability to run large-scale screens in iPSC-derived myeloid model systems.
2025-04-30 | GSE289724 | GEO
Project description:Recent advances in functional genomics tools have ushered in a new era of genetic editing to identify molecular pathways relevant to developmental and disease biology. However, limited model systems are available that adequately mimic cell states and phenotypes associated with human disease pathways. Here, we quantitatively analyzed the founder population bottleneck effect and demonstrated how the population changes from induced pluripotent stem cells (iPSCs) to hematopoietic stem cells and to the final induced macrophage population. We then engineered SAMHD1 knockout (KO) iPSC and characterized the iPSC line with RNA Seq, and induced macrophages from two distinct protocols with functional analysis. We then generated SAMHD1 KO CRISPR-dCAS9 KRAB iPSC through lenti-viral transduction aiming to increase the efficiency of lentiviral mediated gene transfer. We demonstrated increased lenti-viral transduction efficiency in induced macrophage, as well as microglia induced with two distinct protocols. This model allows for efficient gene knock down, as well as large-scale functional genomics screens in mature iPSC-derived macrophages or microglia with applications in innate immunity and chronic inflammatory disease biology. These experiments highlight the broad applicability of this platform for disease-relevant target identification and may improve our ability to run large-scale screens in iPSC-derived myeloid model systems.
2025-04-30 | GSE289725 | GEO
Project description:SAMHD1 Knockout iPSC model enables high lenti-viral transduction in myeloid cell types [barcode_count]
| PRJNA1224259 | ENA
Project description:To investigate the contribution of type-1 IFN signalling to the upregulation of IFN- stimulated genes in SAMHD1-deficient cells, we performed global gene expression analysis of SAMHD1-deficient IFNAR-/- macrophages. Peritoneal macrophages from ten SAMHD1-deficient IFNAR-/- and six SAMHD1-deficient controls were FACS sorted. RNA was subjected to next generation mRNA sequencing.
2013-07-01 | E-GEOD-45358 | biostudies-arrayexpress
Project description:SAMHD1 restricts HIV-1 replication in dendritic and other myeloid cells. SAMHD1 has been shown to possess a dGTP-dependent dNTP triphosphatase (dNTPase) activity and is proposed to inhibit HIV-1 replication by depleting the intracellular dNTP pool. Arguing against a role for SAMHD1 dNTPase in HIV-1 restriction, the phosphorylation of SAMHD1 regulates the restriction activity toward HIV-1 without affecting its ability to decrease cellular dNTP levels. Here, we show that SAMHD1 is a phospho-regulated RNase and that the RNase function is required for HIV-1 restriction. Mutation of the SAMHD1 D137 residue in the allosteric site (SAMHD1D137N) abolishes dNTPase activity but has no effect on RNase activity. This dNTPase-defective SAMHD1D137N mutant is able to restrict HIV-1 infection to nearly the same extent as wild-type SAMHD1. SAMHD1 associates with and degrades the HIV-1 genomic RNA during the early phases of infection. SAMHD1 silencing in macrophages and CD4+ T cells from healthy donors increases HIV-1 RNA stability, thus rendering the cells permissive for HIV-1 infection. Furthermore, the phosphorylation of SAMHD1 at position T592 abolishes the RNase activity toward HIV-1 RNA, and consequently the ability of SAMHD1 to restrict HIV-1 infection, uncovering the phosphorylation of SAMHD1 T592 as a negative regulatory mechanism of RNase activity. Together, our results demonstrate that SAMHD1 is an essential RNase that prevents HIV-1 infection by directly degrading HIV-1 genomic RNA in a phosphorylation-regulated manner. The unique property of SAMHD1 that cleaves HIV-1 genomic RNA with no sequence preferences could be exploited to develop a new class of intervention for error-prone retroviruses.
2014-06-13 | GSE58418 | GEO
Project description:We identified novel recurrent genetic lesions in T-PLL affecting genes involved in JAK/STAT signaling (PTPRC), epigenetic regulation (PRDM2), or DNA damage repair (SAMHD1, PARP10, HERC1, HERC2). Mutations of the tumor suppressor gene SAMHD1 causing amino-acid exchanges or protein truncations as well as copy number variations in SAMHD1 were seen in 20% of cases.
2018-07-23 | GSE100844 | GEO
Project description:To elucidate responses of myeloid cells to SAMHD1 deficiency in the absence of exogenous viral infection, we performed global gene expression analysis of SAMHD1 deficient macrophages.
2012-12-01 | GSE37236 | GEO
Project description:To elucidate responses of myeloid cells to SAMHD1 deficiency in the absence of exogenous viral infection, we performed global gene expression analysis of SAMHD1 deficient macrophages. Peritoneal macrophages from nine mutants and nine controls were FACS sorted. Cells from three animals were pooled to yield three poolls per group. RNA from these pools was subjected to next generation mRNA sequencing
2012-12-01 | E-GEOD-37236 | biostudies-arrayexpress
Project description:We identified novel recurrent genetic lesions in T-PLL affecting genes involved in JAK/STAT signaling (PTPRC), epigenetic regulation (PRDM2), or DNA damage repair (SAMHD1, PARP10, HERC1, HERC2). Mutations of the tumor suppressor gene SAMHD1 causing amino-acid exchanges or protein truncations as well as copy number variations in SAMHD1 were seen in 20% of cases.
2018-02-22 | GSE100882 | GEO
Project description:In mammalian cells, the catabolic activity of the dNTP triphosphohydrolase SAMHD1 sets the balance and the concentrations of the four dNTPs. Deficiency of SAMHD1 leads to unequally increased pools and marked dNTP imbalance. Although it is documented that imbalanced dNTP pool expansion increases mutation frequency in cancer cells, it is not known if the SAMHD1-induced dNTP imbalance favors accumulation of somatic mutations in non-transformed cells. Here we have investigated how fibroblasts isolated from Aicardi Goutières Syndrome (AGS) patients with mutated SAMHD1 react to the constitutive pool imbalance characterized by a huge dGTP pool. We focused on the effects on dNTP pools, cell-cycle progression, dynamics and fidelity of DNA replication, efficiency of UV-induced DNA repair. AGS fibroblasts entered senescence prematurely or upregulated genes involved in G1/S transition and DNA replication. The normally growing AGS cells exhibited unchanged DNA replication dynamics and, when quiescent, faster rate of excision repair of UV-induced DNA damages than wildtype fibroblasts. To investigate if the lack of SAMHD1 affects DNA replication fidelity we compared de novo mutations in AGS and WT cells by exome next generation sequencing. Somatic variant analysis indicated a mutator phenotype suggesting that SAMHD1 is a caretaker gene whose deficiency is per se mutagenic promoting genome instability in non-transformed cells.
2019-12-03 | GSE135652 | GEO