rosa26 targeting vector construct ai9  (Addgene inc)

Journal: Nature Communications

Article Title: Editing DNA methylation in vivo

doi: 10.1038/s41467-025-67222-5

Figure Lengend Snippet: A Schematic representation of the Lox-Stop-Lox-dCas9-DNMT3A-P2A-GFP (LSL-dC9-D) transgene cassette inserted at the Rosa26 locus. pCAG cytomegalovirus enhancer fused with chicken beta-actin promoter and rabbit beta-globin splice acceptor, LSL Lox-stop-lox cassette, NLS nuclear localization sequence, P2A porcine teschivoris-1 2A self-cleaving sequence, eGFP enhanced green fluorescent protein, WPRE woodchuck hepatitis virus posttranscriptional regulatory element, bGHpA bovine growth hormone polyadenylation signal. B Western blot of DNMT3A, dCas9, and Tubulin expressions in brain tissue isolated from LSL-dCas9-DNMT3A-GFP mice and LSL-dCas9-DNMT3A-GFP; EIIa-Cre mice. C Immunofluorescent staining of GFP in the hippocampus of LSL-dCas9-DNMT3A-GFP and LSL-dCas9-DNMT3A-GFP; EIIa-Cre mice. Scale bar: 100 μm. D Immunofluorescent staining of DAPI, mCherry, GFP, dCas9 colocalization in mice injected contralaterally with either AAV9-mCherry or AAV9-mCherry-Cre. Scale bar: 100 μm. E Quantification of dCas9-DNMT3A induction efficiency in mCherry and mCherry-Cre labeled cells. ( n = 3 mice per group, two-sided t test, P = 0.000010).

Article Snippet: The targeted KV-1 mESC clones were generated by electroporation of the modified Rosa26 targeting vector construct Ai9 (Addgene 22799) containing a transgenic LSL-dCas9-DNMT3A or LSL-dCas9-TET1 cassette and identification of positive clones by genotyping PCR.

Techniques: Sequencing, Virus, Western Blot, Isolation, Staining, Injection, Labeling

Journal: Nature Communications

Article Title: Editing DNA methylation in vivo

doi: 10.1038/s41467-025-67222-5

Figure Lengend Snippet: A Schematic representation of the Lox-Stop-Lox-dCas9-TET1-P2A-GFP (LSL-dC-T) transgene cassette inserted at the Rosa26 locus. pCAG cytomegalovirus enhancer fused with chicken beta-actin promoter and rabbit beta-globin splice acceptor, LSL Lox-stop-lox cassette, NLS nuclear localization sequence, P2A porcine teschivoris-1 2A self-cleaving sequence, eGFP enhanced green fluorescent protein, WPRE woodchuck hepatitis virus posttranscriptional regulatory element, bGHpA bovine growth hormone polyadenylation signal. B Western blot of GFP, dCas9, and Tubulin expressions in brain tissue isolated from LSL-dCas9-TET1-GFP mice and LSL-dCas9-TET1-GFP; EIIa-Cre mice. C Immunofluorescent staining of GFP in the hippocampus of LSL-dCas9-TET1-GFP and LSL-dCas9-TET1-GFP; EIIa-Cre mice. Scale bar: 100 μm. D Immunofluorescent staining of DAPI, mCherry, eGFP, dCas9 colocalization in mice injected contralaterally with either mCherry or mCherry-Cre. Scale bar: 100 μm. E Quantification of dCas9-TET1 induction efficiency in mCherry-Cre and mCherry-labeled cells. ( n = 3 mice per group, two-sided t test, P = 1.2 × 10 −7 ). F Quantification of the percentage of NeuN+ cells in mCherry− and mCherry+ populations. ( n = 8 mice per group, two-sided t test, P = 2.81 × 10 −8 ).

Article Snippet: The targeted KV-1 mESC clones were generated by electroporation of the modified Rosa26 targeting vector construct Ai9 (Addgene 22799) containing a transgenic LSL-dCas9-DNMT3A or LSL-dCas9-TET1 cassette and identification of positive clones by genotyping PCR.

Techniques: Sequencing, Virus, Western Blot, Isolation, Staining, Injection, Labeling

Journal: Research Square

Article Title: Expression of the CIC-DUX4 fusion oncoprotein mimics human CIC-rearranged sarcoma in genetically engineered mouse models

doi: 10.21203/rs.3.rs-3487637/v1

Figure Lengend Snippet: CIC haploinsufficiency is not required for CIC-DUX4 sarcomagenesis. a) Schematic of the Ai9CDS allele; Cre-loxP recombination removes a stop cassette and permits expression of an HA-CIC-DUX4 fusion gene at the Rosa26 locus. b) Kaplan-Meier survival curve of chimeric Ai9CDS animals. c) DNA gel of amplification products from PCR across the loxP sites in tails, tumors, and tumor-derived cell lines (n=2 each). d) Schematic of the TOPCDS allele; Cre-loxP recombination removes a stop cassette (containing multiple polyA sequences in tandem) and permits expression of an HA-CIC-DUX4 fusion gene at the Rosa26 locus. e) Kaplan-Meier survival curve of chimeric TOPCDS animals. f) DNA gel of amplification products from PCR across the loxP sites in tails, tumors, and tumor-derived cell lines (n=2 each).

Article Snippet: Rosa26 Lox-STOP-Lox (LSL) Ai9-HA-CIC-DUX4 (R26-Ai9) mice were generated by subcloning an N-terminal 3x HA-tagged CIC-DUX4 fusion gene from Yoshimoto et al. into a Rosa26 targeting construct (Addgene #21714).

Techniques: Expressing, Amplification, Derivative Assay