Biology subjects

Van den akker, E.

Publications and source records attributed to Van den akker, E..

3 recordsLinked to original sources

bioRxiv2020

Highly efficient genome editing in hematopoietic stem and progenitor cells

Ribonucleoproteins (RNPs) are frequently applied for therapeutic gene editing as well as fundamental research, because the method is fast, viral free, and does not rely on clonal selection. We evaluated various parameters to genetically engineer human hematopoietic stem progenitor cells (HSPCs) using spCas9-RNPs and achieve gene editing efficiencies up to 80%. We find that single guide RNA (sgRNA) design is critical to achieve high gene editing efficiencies. However, finding effective sgRNAs for HSPCs can be challenging, while the contribution of numerous in silico models is unclear. Here we established a time- and cost-efficient in vitro transcribed sgRNA screening model in K562 cells to identify sgRNAs that are effective in HSPCs using RNP delivery. We show that this simple screening method outperforms all in silico prediction models. Our data demonstrates that most in silico sgRNA prediction models are ineffective and we make recommendations to potentially improve their accuracy. We report that gene editing is equally efficient in distinct CD34+ HSPC subpopulations. Furthermore, no effects on cell proliferation, differentiation or in vitro hematopoietic lineage commitment were observed. Finally, no upregulation of p21 expression was found, suggesting unperturbed HSPC homeostasis. Key pointsO_LIIn vitro transcribed single sgRNAs (IVTsgRNA) screening in K562 outperforms in silico modeling C_LIO_LIHematopoietic stem and progenitor cells are equally targeted by Ribonucleoproteins (RNPs) C_LIO_LIHematopoietic stem and progenitor cells show no induction of p21 expression or effects on differentiation, proliferation and lineage commitment C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=158 SRC="FIGDIR/small/423329v2_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@279812org.highwire.dtl.DTLVardef@170fe3dorg.highwire.dtl.DTLVardef@1438cdforg.highwire.dtl.DTLVardef@1d5c2ab_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2020

Abundant circular RNA expression in terminal myeloid differentiation does not correlate with frequent circRNA translation, or with circRNA-mediated translation regulation

Each day, about 1012 erythrocytes and platelets are released into the blood stream. This substantial output from hematopoietic stem cells is tightly regulated by transcriptional and epigenetic factors. Whether and how circular RNAs (circRNAs) contribute to the differentiation and/or identity of hematopoietic cells is to date not known. We recently reported that erythrocytes and platelets contain the highest levels and numbers of circRNAs amongst hematopoietic cells. Here, we provide the first detailed analysis of circRNA expression during erythroid and megakaryoid differentiation. CircRNA expression not only significantly increased upon enucleation, but also had limited overlap between progenitor cells and mature cells, suggesting that circRNA expression stems from regulated processes rather than resulting from mere accumulation. To study circRNA function in hematopoiesis, we first compared the expression levels of circRNAs with the translation efficiency of their mRNA-counterpart. We found that only 1 out of 2531 (0.04%) circRNAs associated with mRNA-translation regulation. Furthermore, irrespective of 1000s of identified putative open reading frames, deep ribosome-footprinting sequencing and mass spectrometry analysis provided little evidence for translation of endogenously expressed circRNAs. In conclusion, circRNAs alter their expression profile during terminal hematopoietic differentiation, yet their contribution to regulate cellular processes remains enigmatic.

molecular biology↗

bioRxiv2020

Mouse Classical and Non-Classical Monocytes Express Comparable Levels of Chemokine Receptor CX3CR1

Human and mouse monocytes are divided into two subpopulations, classical (C-Mo) and non-classical (NC-Mo) monocytes. CC-chemokine receptor 2 (CCR2) and CX3C-chemokine receptor 1 (CX3CR1) are common features of monocyte subsets between humans and mice, i.e., C-Mo and NC-Mo are characterized as CCR2highCX3CR1low and CCR2lowCX3CR1high. Since many studies utilize mouse models to investigate roles of monocytes in human diseases, it is important to understand the similarities and differences between human and mouse monocytic subsets. In this study, we show that the expression of Cx3cr1 mRNA and CX3CR1 cell surface protein are different between circulating monocytic subsets in human but not in mice. We analyzed monocyte subsets in the blood using wild type C57BL/6 and Cx3cr1-GFP knock-in (Cx3cr1GFP/+) reporter mice. We observed higher Cx3cr1 promoter activity indicated by GFP expression in NC-Mo compared to C-Mo. However, there were no differences between the subsets in CX3CR1 mRNA nor surface protein expression determined by anti-CX3CR1 antibody or binding of fluorophore-conjugated ligand. However in the bone marrow of Cx3cr1GFP/+ mice, CX3CR1 expression was higher in NC-Mo compared to C-Mo, suggesting that mouse NC-Mo express higher level of CX3CR1 than C-Mo in the bone but this difference disappears in the blood. In contrast, human NC-Mo differentially expressed CX3CR1 compared to C-Mo in both blood and bone marrow. Given these findings, the discrepancy between promoter activity and protein levels should be considered when the roles of CX3CR1 are investigated in mouse models of human diseases. Summary sentenceIn this study, we show that the expression of Cx3cr1 mRNA is different between circulating monocytic subsets in human but not in mice.

immunology↗