Article Title: LUBAC is essential for embryogenesis by preventing cell death and enabling haematopoiesis
Figure Lengend Snippet: HOIL-1-deficient mice die at mid-gestation a, Schematic representation of the Hoil-1 knockout strategy. Solid boxes represent Hoil-1 exons and grey boxes with a star indicate the targeted exons. Boxes with diagonal and horizontal strips represent LoxP and Frt sites, respectively. b, Specificity of gene recombination was assessed by Southern blotting with 5’ and 3’ probes external to the construct in four clones (14B8, 14F6, 20D7 and 21F7). Digest of the DNA with ApaI, followed by hybridisation with the 3’ probe was expected to show a 5700 bp band for the WT allele and a 7700 bp band for the mutant allele. All four clones appeared to have the correct recombination on the 3’ side. Digest of the DNA with SphI and hybridisation with the 5’ probe was expected to show a 4500 bp WT band and a 6200 bp band for the mutated allele. Clones 14B8, 14F6 and 21F7 appeared to be correctly recombined on the 5’ side. Finally, cutting the DNA with ApaI and hybridising with a hygro probe showed a single band in all clones, indicative of a single integration of the construct in all four ES clones. Clones 14B8 and 14F6 were selected for generation of the two Hoil-1 -/- strains. c , PCR analysis of Hoil-1 wild-type, heterozygous and knockout mice. d , Protein levels of HOIL-1, HOIP and SHARPIN in whole embryo lysates ( n = 3 for Hoil-1 +/- and Hoil-1 -/- embryos and n =1 for Hoil-1 +/+ . e , Quantification of genotypes of animals obtained from inter-crossing C20Hoil-1 +/- mice. *indicates dead embryos. f , Representative images of C20Hoil-1 +/- and C20Hoil-1 -/- embryos from E9.5 to E11.5 as quantified in (e). Scale bar: 2 mm. g, Single staining showing vascularisation (PECAM-1, top panel) and apoptosis (cleaved Caspase-3, bottom panel) of yolk sacs. Merged image is shown in . h , Whole-mount TUNEL staining of embryos at the indicated stages (embryo/genotype n =2 at E10.5, n =8 for Hoil-1 +/- and n =5 for Hoil-1 -/- at E11.5). Scale bar: 2 mm. i, Quantification of genotypes of animals obtained from inter-crossing Hoil-1 fl/wt Tie2-Cre + with Hoil-1 fl/fl Tie2-Cre - mice. *indicates dead embryos. j , Representative images of embryos with conditional deletion of Hoil-1 in Tie2-Cre expressing cells as quantified in (i). Scale bar: 2 mm. *: poorly vascularised yolk sac. Fig. 1c
Article Snippet: Yolk sacs were stained with antibodies against PECAM-1 (BD Biosciences, 5533370 Clone MEC13.3) and cleaved caspase-3 (Cell Signaling, 9664), followed by staining with secondary antibodies, Alexa Fluor 594 Goat anti-Rat IgG and Alexa Fluor 488 Goat anti-Rabbit IgG (Invitrogen, A-11007 and A-11034, respectively), and analysed by fluorescent microscopy.
Techniques: Mouse Assay, Knock-Out, Southern Blot, Construct, Clone Assay, Hybridization, Mutagenesis, Polymerase Chain Reaction, Staining, TUNEL Assay, Expressing