CRISPR/Cas9 editing in conditionally immortalized HoxB8 cells for studying gene regulation in mouse dendritic cells

Xu, Huaming; Look, Thomas; Prithiviraj, Sujeethkumar; Lennartz, Daniel; Cáceres, Manuel Delgado; Götz, Katrin; Wanek, Paul; Kramann, Rafael; Seré, Kristin; Zenke, Martin

doi:10.1002/eji.202149482

Cited by 10 publications

(35 citation statements)

References 11 publications

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“…In the future, it will be of interest to determine conditions that induce functional reprogramming of YS-derived macrophages as well as its impact on organ functions or priming towards the development of inflammatory conditions. In line with previous work using Hoxb8-mediated conditional immortalization, these macrophages will enable further studies into their biological functions as well as their genetic modifications [ 47 , 48 ].…”

Section: Discussionmentioning

confidence: 54%

Differences in Cell-Intrinsic Inflammatory Programs of Yolk Sac and Bone Marrow Macrophages

Elhag

Stremmel

Zehrer

et al. 2021

Cells

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Background: Tissue-resident macrophages have mixed developmental origins. They derive in variable extent from yolk sac (YS) hematopoiesis during embryonic development. Bone marrow (BM) hematopoietic progenitors give rise to tissue macrophages in postnatal life, and their contribution increases upon organ injury. Since the phenotype and functions of macrophages are modulated by the tissue of residence, the impact of their origin and developmental paths has remained incompletely understood. Methods: In order to decipher cell-intrinsic macrophage programs, we immortalized hematopoietic progenitors from YS and BM using conditional HoxB8, and carried out an in-depth functional and molecular analysis of differentiated macrophages. Results: While YS and BM macrophages demonstrate close similarities in terms of cellular growth, differentiation, cell death susceptibility and phagocytic properties, they display differences in cell metabolism, expression of inflammatory markers and inflammasome activation. Reduced abundance of PYCARD (ASC) and CASPASE-1 proteins in YS macrophages abrogated interleukin-1β production in response to canonical and non-canonical inflammasome activation. Conclusions: Macrophage ontogeny is associated with distinct cellular programs and immune response. Our findings contribute to the understanding of the regulation and programming of macrophage functions.

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Section: Discussionmentioning

confidence: 54%

Differences in Cell-Intrinsic Inflammatory Programs of Yolk Sac and Bone Marrow Macrophages

Elhag

Stremmel

Zehrer

et al. 2021

Cells

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“…HoxB8 MPP were obtained from bone marrow of Mx-Cas9-GFP mice by infection with the estrogen (E2) inducible HoxB8-ER. These HoxB9 MPP exhibited an extended lifespan and robust clonogenic potential and differentiated into all DC subsets in vitro and in vivo (Xu et al, 2021). Infection of gRNA targeting lncIRF8 promoter in Mx-Cas9-GFP HoxB8 MPP and induction of Cas9 by interferon generated single-cell lncIRF8 promoter KO clones.…”

Section: Resultsmentioning

confidence: 99%

“…Generation of a precise deletion requires clonal cell populations, which is hardly achieved in somatic cells due to their limited life span. Therefore, we developed a Mx-Cas9-GFP system of conditionally immortalized HoxB8 MPP, which upon differentiation faithfully recapitulate DC development (Figure S3A and B) (Xu et al, 2021). HoxB8 MPP were obtained from bone marrow of Mx-Cas9-GFP mice by infection with the estrogen (E2) inducible HoxB8-ER.…”

Section: Lncirf8 Promoter Ko Compromises Pdc and Cdc1 Developmentmentioning

confidence: 99%

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A lncRNA identifiesIRF8enhancer element in negative feedback control of dendritic cell differentiation

Kuo

et al. 2022

Preprint

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Transcription factors play a determining role in lineage commitment and cell differentiation. Interferon regulatory factor 8 (IRF8), a hematopoietic transcription factor, is prominently upregulated in dendritic cells (DC) by autoactivation and controls DC differentiation and function. However, it is unclear how IRF8 autoactivation is controlled and eventually limited. Here we identified a novel long non-coding RNA transcribed from the +32 kb enhancer downstream of IRF8 transcription start site and expressed specifically in plasmacytoid DC (pDC), referred to as lncIRF8. A sequence element of the lncIRF8 promoter, but not lncIRF8 itself, is crucial for pDC and classical DC type 1 (cDC1) differentiation. In DC development IRF8 autoactivation is first initiated by flanking enhancers and then second controlled by feedback inhibition through the lncIRF8 promoter element in the +32 kb enhancer. Our work reveals a previously unrecognized negative feedback loop of IRF8 that orchestrates its own expression and thereby controls DC differentiation.

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“…Current Protocols 2018; Leithner et al, 2018;Orosz, Walzog, & Mócsai, 2020;Redecke et al, 2013;Xu et al, 2021).…”

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Quantifying the Probing and Selection of Microenvironmental Pores by Motile Immune Cells

Kroll¹,

Ruiz‐Fernandez²,

Braun³

et al. 2022

Current Protocols

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Immune cells are constantly on the move through multicellular organisms to explore and respond to pathogens and other harmful insults. While moving, immune cells efficiently traverse microenvironments composed of tissue cells and extracellular fibers, which together form complex environments of various porosity, stiffness, topography, and chemical composition. In this protocol we describe experimental procedures to investigate immune cell migration through microenvironments of heterogeneous porosity. In particular, we describe micro‐channels, micro‐pillars, and collagen networks as cell migration paths with alternative pore size choices. Employing micro‐channels or micro‐pillars that divide at junctions into alternative paths with initially differentially sized pores allows us to precisely (1) measure the cellular translocation time through these porous path junctions, (2) quantify the cellular preference for individual pore sizes, and (3) image cellular components like the nucleus and the cytoskeleton. This reductionistic experimental setup thus can elucidate how immune cells perform decisions in complex microenvironments of various porosity like the interstitium. The setup further allows investigation of the underlying forces of cellular squeezing and the consequences of cellular deformation on the integrity of the cell and its organelles. As a complementary approach that does not require any micro‐engineering expertise, we describe the usage of three‐dimensional collagen networks with different pore sizes. Whereas we here focus on dendritic cells as a model for motile immune cells, the described protocols are versatile as they are also applicable for other immune cell types like neutrophils and non‐immune cell types such as mesenchymal and cancer cells. In summary, we here describe protocols to identify the mechanisms and principles of cellular probing, decision making, and squeezing during cellular movement through microenvironments of heterogeneous porosity. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Immune cell migration in micro‐channels and micro‐pillars with defined pore sizes Support Protocol 1: Epoxy replica of generated and/or published micro‐structures Support Protocol 2: Dendritic cell differentiation Basic Protocol 2: Immune cell migration in 3D collagen networks of variable pore sizes

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CRISPR/Cas9 editing in conditionally immortalized HoxB8 cells for studying gene regulation in mouse dendritic cells

Cited by 10 publications

References 11 publications

Differences in Cell-Intrinsic Inflammatory Programs of Yolk Sac and Bone Marrow Macrophages

Differences in Cell-Intrinsic Inflammatory Programs of Yolk Sac and Bone Marrow Macrophages

A lncRNA identifiesIRF8enhancer element in negative feedback control of dendritic cell differentiation

Quantifying the Probing and Selection of Microenvironmental Pores by Motile Immune Cells

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