u6 promoter driven grna expression cassette  (Addgene inc)

Journal: Nucleic Acids Research

Article Title: Comparison of TALE designer transcription factors and the CRISPR/dCas9 in regulation of gene expression by targeting enhancers

doi: 10.1093/nar/gku836

Figure Lengend Snippet: Activation of the mouse Oct4 and Nanog loci by TALE-As and dCas9-As/gRNAs. ( A ) Schematic diagram of the dCas9-As evaluated in this study. In all cases, blue florescent protein (BFP) was used to track the expression of dCas9-As. gRNA expression is controlled by U6 promoter, and EF1a-mCherry is used to detect the integration of the vector into the genome. A blasticidin resistance and the reverse tetracycline transactivator (rtTA) cassettes were also linked to the mCherry cassette by T2A peptides. PB-5TR and PB-3TR are the two terminal repeat sequences of the piggyBac (PB) transposon. ( B ) Schematic diagram of the TALE and dCas9/gRNA targeting sites at the mouse Oct4 and Nanog enhancers. Red arrows indicate the gRNA targeting sites and the blue arrows mark the TALE sites. ( C ) Activation of the Oct4 distal enhancer luciferase reporter by the TALE-As and dCas9-As/gRNAs. ( D ) qRT-PCR analysis of the Oct4 mRNA levels in MEFs expressing the TALE-As or dCas9-As/gRNAs. ( E ) Activation of the Nanog enhancer luciferase reporter by the TALE-As and dCas9-As/gRNAs. ( F ) qRT-PCR analysis of the Nanog mRNA levels in EpiSCs expressing the TALE-As or dCas9-As/gRNAs. All gene expression values were normalized to Gapdh . Results were representative of three independent experiments and were presented as ±SD, n = 3. * P < 0.05.

Article Snippet: To generate the PB-U6-gRNA–EFα-mCherry-2A-rtTA-2A-BSD vector, the U6 promoter driven gRNA expression cassette (Addgene 44248) was first cloned into the PB-LTR vector.

Techniques: Activation Assay, Expressing, Plasmid Preparation, Luciferase, Quantitative RT-PCR, Gene Expression

Journal: Nucleic Acids Research

Article Title: Comparison of TALE designer transcription factors and the CRISPR/dCas9 in regulation of gene expression by targeting enhancers

doi: 10.1093/nar/gku836

Figure Lengend Snippet: Repression of the Oct4 and Nanog loci by TALE-Rs and dCas9-Rs/gRNAs. ( A ) Schematic diagram of the dCas9-R designs evaluated in this study. In all cases, blue florescent protein (BFP) was used to track the expression of dCas9-R. The gRNA vector used was the same as described in Figure . ( B ) Repression of the endogenous Oct4 locus in Oct4-GFP ES cells indicated by the reduction of GFP intensity in flow cytometric analysis on day 0 and day 3 of expression of the TALE-Rs or dCas9-Rs/gRNAs targeting at the Sites 1–4 of the Oct4 distal enhancer (Gated mCherry + for TALEs and mCherry + /BFP + for dCas9-As/gRNAs). ( C ) Comparison of Oct4 expression levels in Oct4-GFP ES cells expressing the TALE-Rs or dCas9-Rs targeting at the Site 1 or Site 3 of the Oct4 distal enhancer by qRT-PCR. ( D ) The repressive effect of TALE-R and dCas9-Rs/gRNAs targeting at the Site 3 of the Oct4 distal enhancer on MEF reprogramming. MEFs were reprogrammed with Dox inducible CKS and Lrh1 (CKSL) factors together with the TALE-R or dCas9-R/gRNAs as in (C). ‘CKSL+’ indicates that all transfections have CKSL. ‘−’ is the CKSL only control (no repressor). ‘CKS only’ is the reprogramming negative control. ( E ) Reprogramming using FACS-sorted MEFs (as described in 2E) to control transfection variability. Wild-type MEFs were transfected and sorted on day 2 for GFP + /mCherry + in the TALE-R transfection and for GFP + /mCherry + /BFP + in dCas9-R/gRNAs (PL-R) experiments. Both TALE-Rs and dCas9-R/gRNAs (PL-R) targeted the Site 2–4 of the Oct4 distal enhancer. Sorted MEFs were re-plated (20 000 cells/ well) for reprogramming and iPSC colonies were scored by AP staining. ( F ) The repressive effect of TALE-Rs or dCas9-Rs/gRNAs targeting at the Sites 1–2 of the Nanog 5 kb enhancer in Nanog-GFP ES cells. Site 1 is located outside the enhancer region whereas Site 2 is inside. The Nanog repression was demonstrated by the increase of the GFP low/dim fraction in Nanog-GFP ES cells. ( G ) Endogenous N anog mRNA levels in Nanog-GFP ES cells expressing the TALE-Rs or dCas9-Rs targeting at the Sites 1–2. ( H ) Repression of Klf4- mediated EpiSC reprogramming to iPSCs by the TALE-R and dCas9-Rs targeting at the Site 2 of the Nanog 5 kb enhancer. ‘Klf4+’ refers to the transfections combined with Klf4 and ‘−’ is the Klf4 control (no effector). Results were representative of three independent experiments and were presented as ±SD, n = 3.

Article Snippet: To generate the PB-U6-gRNA–EFα-mCherry-2A-rtTA-2A-BSD vector, the U6 promoter driven gRNA expression cassette (Addgene 44248) was first cloned into the PB-LTR vector.

Techniques: Expressing, Plasmid Preparation, Comparison, Quantitative RT-PCR, Transfection, Control, Negative Control, Staining