Journal: Nature Communications
Article Title: Interferon priming is essential for human CD34+ cell-derived plasmacytoid dendritic cell maturation and function
Figure Lengend Snippet: HSPC-pDCs are amenable to genetic modifications. a Graphical illustration of the protocol for CRISPR/Cas9-mediated gene editing using Cas9 RNP. CD34 + HSPCs were initially cultured in CD34 + HSPC medium at low density for 3 days before being electroporated with Cas9 RNP complexes. After a 3-day recovery phase, pDC differentiation was initiated. b Expansion of cells during differentiation from HSPCs into HSPC-pDCs. c Indel frequencies after the 3-day recovery phase quantified by TIDE analysis for cells targeted at MyD88 , IFNAR1 , and CCR5 (control). d Numbers of HSPC-pDCs at day 21 for gene-edited HSPC-pDCs. e Western blot analysis showing induction of pSTAT1 upon 3 h IFN-β stimulation (upper panel) and protein levels of MyD88 (lower panel) in HSPC-pDCs targeted at IFNAR1 and MyD88 , respectively (detection Ab for STAT1: CST 9172). Indel frequencies at the respective targets are listed below the western blot. f , g Surface expression levels (MFI) of CD123 ( f ) and CD304 ( g ) in IFN-primed and unprimed HSPC-pDCs with gene editing at MyD88 , IFNAR1, and CCR5 . h Functional levels of type I IFN after stimulation with R837 (TLR7) or CpG-A (TLR9) in unprimed and IFN-primed HSPC-pDCs gene-edited at CCR5 , MyD88 , or CCR5 . i Levels of IL-6 after stimulation with R837 (TLR7) or CpG-A (TLR9) in unprimed and IFN-primed HSPC-pDCs targeted at CCR5, MyD88, or IFNAR1 . Data are ±SEM of five ( b – d ) or three ( f – i
Article Snippet: Ribonucleoprotein (RNP) complexes were made by incubating Cas9 protein (Integrated DNA Technologies) with sgRNA at a molar ratio of 1:2.5 at 25 °C for 10 min prior to nucleoporation.
Techniques: CRISPR, Cell Culture, Western Blot, Expressing, Functional Assay