Journal: bioRxiv

Article Title: CTCF Mediates Dosage and Sequence-context-dependent Transcriptional Insulation through Formation of Local Chromatin Domains

doi: 10.1101/2020.07.07.192526

Figure Lengend Snippet: a , Left, the regulatory landscape of the Sox2 locus in mES cells. Orientations of CTCF sites are indicated on the top of the signal tracks; Right, genetic constructs of mES cell lines. Boxed Sox2 in green represents Sox2-p2a-egfp in situ fusion gene, boxed Sox2 in red represents Sox2-p2a-mCherry in situ fusion gene. The hygromycin phosphotransferase-thymidine kinase fusion gene HyTK is flanked by Flippase recognition sites FRT and F3 . b , Experimental scheme to insert a test sequence into the Sox2 locus by recombinase mediated cassette exchange (RMCE). The Flippase expression plasmid and donor plasmid containing the test sequence were co-electroporated into cells. The donor plasmid contains Not1 and Sbf1 restriction enzyme sites so that the orientation of the insert can be controlled. Mouse ES cell clones containing the insert were picked, genotyped, and allelic Sox2 expression was measured by FACS. c , A bar graph shows the normalized Sox2-eGFP expression of the no insertion clone (n=8), different CBS insertion clones (n=3. For Sox9_CBS1 in forward orientation, n=2.) and downstream insertion controls (n=27). Each dot represents an independently picked colony. One-way analysis of variance with Bonferroni’s multiple comparisons test. ns P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Data are mean ± sd.

Article Snippet: A Flippase expression plasmid(pFlpe) (addgene #13787) and a donor plasmid(pDonor) were co-electroporated into 0.1 million insulator reporter or control cells at the ratio of 1:4 (pFlpe: pDonor = 1μg :4μg).

Techniques: Construct, In Situ, Sequencing, Expressing, Plasmid Preparation, Clone Assay

Journal: bioRxiv

Article Title: CTCF Mediates Dosage and Sequence-context-dependent Transcriptional Insulation through Formation of Local Chromatin Domains

doi: 10.1101/2020.07.07.192526

Figure Lengend Snippet: a , Diagram of recombinase mediated cassette exchange (RMCE) in the insulator reporter cell line. Flippase expression plasmid and the donor plasmid carrying the insertion sequence were co-electroporated into cells. The replacement only happens on the CAST allele. b , Genotyping insertion clones of λDNA fragments generated by RMCE. PCR primers were designed from genomic locations that spanned the insertion position. Top band, insertion fragment; Bottom band, PCR product from the no insertion allele.

Article Snippet: A Flippase expression plasmid(pFlpe) (addgene #13787) and a donor plasmid(pDonor) were co-electroporated into 0.1 million insulator reporter or control cells at the ratio of 1:4 (pFlpe: pDonor = 1μg :4μg).

Techniques: Expressing, Plasmid Preparation, Sequencing, Clone Assay, Generated

Journal: Frontiers in Immunology

Article Title: ISR8/IRF1-AS1 Is Relevant for IFNα and NF-κB Responses

doi: 10.3389/fimmu.2022.829335

Figure Lengend Snippet: Analysis of the response of episomic or genomic reporter genes in ISR8 -disrupted cells after IRF1 or RELA overexpression and IFNα treatment. (A) Evaluation of relative luciferase units (RLU) in the indicated cells transfected for 48 h with pISRE-Luc and treated with the indicated doses of IFNα. A plasmid expressing renilla´s luciferase was also co-transfected in all cases as a control. (B) Similar to A but cells were co-transfected with pCMV-Luc, pISRE-Luc and a control plasmid or pIRF1. (C) Similar to C but cells were transfected with pIRF-Luc and pIRF1 or pIRF3. (D) Relative luciferase units (RLU) in the indicated cells transfected with pNF-κB-Luc and mock-treated or treated with TNFα for 6 h. (E) RELA mRNA was evaluated in HeLa and pNISR8 cells by qRT-PCR and normalized to GAPDH mRNA. (F) Schematic of the experiment is shown to the left. The indicated cells were transfected with pSB-IRF·ISRE-Luc and plasmids expressing renilla´s luciferase and the transposase. Forty-eight hours or 9 days after transfection and puromycin selection, luciferase signal was measured and fold change (FC) of RLU in IFNα-treated and untreated cells is shown. Error bars indicate standard deviations. Experiments were performed at least four times and a representative figure is shown.

Article Snippet: The resulting pSBIRF·ISRE-Luc vector was co-transfected with the SB transposase expression vector (Addgene #127909) to generate stable cells.

Techniques: Over Expression, Luciferase, Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Selection