Fig. 4

Genome editing of DE4 enhancer and functional analysis in BAC transgenic mice. a Gene tracks showing genomic locations of downstream enhancers. b CRISPR-mediated deletion of downstream enhancer 4 (DE4) does not affect hA20 expression in 293T cells. qPCR analysis of hA20 mRNA in control and DE4-deleted 293T cells, error bars represent mean ± s.e.m. c Strategy of CRISPR-mediated deletion of DE4 in oocytes from hA20(CTR) mice. d qPCR analysis of hA20 mRNA in B cells, T cells, BMDM, and SF. Primary B cells were isolated from spleens, macrophages cultured from bone marrow (BMDM), and synovial fibroblasts (SFs) were isolated from ankle joints. B cells were stimulated with antibodies against CD40 (2 μg ml⁻¹) and IgM (5 μg ml⁻¹), BMDM with LPS (25 ng ml⁻¹), and SF with TNF (20 ng ml⁻¹) for the indicated times. Pooled data from three independent experiments with three different Δ-DE4 mouse lines is shown. Error bars indicate mean ± s.e.m., statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparison test. e Immunoblot analysis of hA20 protein expression in primary cells obtained and stimulated as in d. Representative of two independent experiments