transduction procedure  (Santa Cruz Biotechnology)

Journal: Tissue Engineering. Part A

Article Title: * CRISPR-Based Epigenome Editing of Cytokine Receptors for the Promotion of Cell Survival and Tissue Deposition in Inflammatory Environments

doi: 10.1089/ten.tea.2016.0441

Figure Lengend Snippet: (A) General mechanism of gene repression by CRISPR-based epigenome editing. Gene expression occurs when chromatin is maintained in the euchromatin (open) configuration by acetylated H3K9 histones. Binding of dCas9-KRAB fusion protein via the single guide RNA (sgRNA) and recognition of the protospacer adjacent (PAM) sequence by dCas9 recruits endogenous factors that replace acetylation of H3K9 with tri-methylation, shifting chromatin to the heterochromatic state, therefore silencing gene expression. (B) Guide RNA sequences for each gene and the control nontarget gRNA sequence with no complementary target. (C) Lentiviral cassette demonstrating components of the epigenome editing system and how their expression is driven. CRISPR, clustered regularly interspaced short palindromic repeats; dCas9, nuclease-deficient Cas9 protein.

Article Snippet: Epigenome editing of inflammatory cytokine receptors in human ADSCs Generation of engineered human ADSCs To generate hADSCs expressing epigenome editing tools and appropriate controls, immortalized hADSCs (SCRC-4000, ATCC) were separately transduced with each of the four lentiviral vectors encoding repressors targeted to the TNFR1 and IL1R1 promoters and the nontarget control vector using lentiviral transduction methods ( Supplementary Materials and Methods ), and cultured in manufacturer recommended expansion media (PCS-500-030, PCS-500-040, ATCC), until analysis.

Techniques: CRISPR, Gene Expression, Binding Assay, Sequencing, Methylation, Control, Expressing

Journal: Tissue Engineering. Part A

Article Title: * CRISPR-Based Epigenome Editing of Cytokine Receptors for the Promotion of Cell Survival and Tissue Deposition in Inflammatory Environments

doi: 10.1089/ten.tea.2016.0441

Figure Lengend Snippet: Verification of lentiviral mediated gene and receptor signaling downregulation in hADSCs. Nontransduced groups abbreviated as “No LV.” (A) TNFR1 and IL1R1 expression in hADSCs post-transduction of epigenome editing system under the control of selected gRNAs (n = 3, * = p < 0.05 [TNFR1/IL1R1-engineered vs. Nontarget control cells]). (B) Fold changes in NF-kB activity post TNF-α/IL-1β dosing in engineered hADSCs that express the most efficient gRNAs (TNFR1: gRNA 1, IL1R1: gRNA 2) and in control cells (n = 4, * = p < 0.05 (cytokine-treated TNFR1/IL1R1-engineered cells vs. cytokine-treated No LV cells), # = p < 0.05 (TNF-α/IL-1β treated cells vs. untreated controls). hADSC, human adipose-derived stem cells.

Article Snippet: Epigenome editing of inflammatory cytokine receptors in human ADSCs Generation of engineered human ADSCs To generate hADSCs expressing epigenome editing tools and appropriate controls, immortalized hADSCs (SCRC-4000, ATCC) were separately transduced with each of the four lentiviral vectors encoding repressors targeted to the TNFR1 and IL1R1 promoters and the nontarget control vector using lentiviral transduction methods ( Supplementary Materials and Methods ), and cultured in manufacturer recommended expansion media (PCS-500-030, PCS-500-040, ATCC), until analysis.

Techniques: Expressing, Transduction, Control, Activity Assay, Derivative Assay