mls mir 17 92 mut92  (Addgene inc)

Journal: Journal of molecular biology

Article Title: The Host miR-17-92 Cluster Negatively Regulates Mouse Mammary Tumor Virus (MMTV) Replication Primarily Via Cluster Member miR-92a.

doi: 10.1016/j.jmb.2024.168738

Figure Lengend Snippet: Figure 2. Over-expression of the miR-17-92 cluster down-regulates MMTV expression. (A) An illustration of the miR-17-92 cluster, its truncated form miR-19a-20a-19b, and miR-92a over-expressed (OE) exogenously in the HEK293T-MMTV cells. (B) GFP-positive stably-transfected cells selected in puromycin and visualized using a fluorescent microscope (10x). (C) Expression of pri-miR-17-92 in miR-17-92 OE cell line quantified using a SYBR Green qPCR assay. (D) TaqMan miRNA assays used to quantify expression of mature miR-17, miR-19a, miR-92a in the miR17-92 OE cell line, (E) miR-19/20 OE cell line, and (F) miR-92a OE cell line. U6 snRNA was used as the endogenous control in these assays. (G) Western blot analysis of MMTV Gag expression in cell lysates. (H) MMTV RNA-specific TaqMan assay to quantify all MMTV messages in the three OE cell lines. (I) MMTV genomic RNA was assessed using gRNA-specific MMTV TaqMan assay in the miR-17-92 cluster OE and the miR-92a OE cells and virus particles isolated from culture supernatants (sup) via ultracentrifugation. The empty vector (EV) was designated as 1 in all analyses. Mean ± SD (n = 3). Statistical significance is shown as * where *p 0.05; **p 0.01, ***p 0.001, ****p 0.0001. (J) Western blot analysis of viral particles harvested from culture supernatants in the respective miRNA OE stable cell lines.

Article Snippet: We would like to thank Prof. Jeffery M. Rosen, Baylor College of Medicine, Houston, TX USA for the gift of HC11 cells, and the Lin He Lab (Lin He, Professor of Cell Biology, Development & Physiology University of California, Berkeley CA, USA) for sharing the miRNA overexpression plasmids: MSCV, MSCV-mir-17-92, MSCV-19a20-19b, MSCV-mir-92, MLS-mir-17-92, MLS- mir17-19b, MLS-mir-17-92-Mut92 (Addgene plasmid #: 24828, 64100, 24827, 64092, 64090, 64089, & 64094).

Techniques: Over Expression, Expressing, Stable Transfection, Transfection, Microscopy, SYBR Green Assay, Control, Western Blot, TaqMan Assay, Virus, Isolation, Plasmid Preparation

Journal: Journal of molecular biology

Article Title: The Host miR-17-92 Cluster Negatively Regulates Mouse Mammary Tumor Virus (MMTV) Replication Primarily Via Cluster Member miR-92a.

doi: 10.1016/j.jmb.2024.168738

Figure Lengend Snippet: Figure 3. Mir-17-92 cluster and miR-92a inhibition rescues MMTV genomic RNA expression. TaqMan miRNA assays were used to quantify: (A) miR-17, miR-19a and miR-92a in miR-17-92 over expression (OE) cell line, (B) miR-19a in the miR-19a-20a-19b OE cell line, and (C) miR-92a in the miR-92a OE cell line. U6 snRNA was used as the endogenous control in the miRNA RT-qPCR assays. The error bars represent ± SD. NC, negative control scrambled oligo specific for each miRNA inhibitor used. (D) Western blot analysis of whole cell lysates (40 mg) from the miRNA OE cell lines treated with miRNA inhibitors for MMTV Gag expression. GAPDH was used as the internal control. Expression of the MMTV genomic RNA was quantified in the: (E) miR-17-92 OE cells treated with a cocktail of anti-miR-17, -19, & -92a oligos, (F) miR-19a- 20a-19b OE cells treated with anti-miR-19a oligos, and (G) miR-92a OE cells treated with anti-miR-92a oligos. b-Actin was used as the internal control in the MMTV TaqMan RT-qPCR assays. The empty vector (EV) was designated as 1 in all analyses. Mean ± SD (n = 3). (H) Western Blot analysis of whole cell lysates (50 mg) from the miR-92a OE cell line treated with anti-miR-92a oligos at varying concentrations. GAPDH was used as the internal control. Statistical significance is shown as * where *p 0.05; **p 0.01, ***p 0.001, ****p 0.0001. ns, not significant; ns (P > 0.05).

Article Snippet: We would like to thank Prof. Jeffery M. Rosen, Baylor College of Medicine, Houston, TX USA for the gift of HC11 cells, and the Lin He Lab (Lin He, Professor of Cell Biology, Development & Physiology University of California, Berkeley CA, USA) for sharing the miRNA overexpression plasmids: MSCV, MSCV-mir-17-92, MSCV-19a20-19b, MSCV-mir-92, MLS-mir-17-92, MLS- mir17-19b, MLS-mir-17-92-Mut92 (Addgene plasmid #: 24828, 64100, 24827, 64092, 64090, 64089, & 64094).

Techniques: Inhibition, RNA Expression, Over Expression, Control, miRNA RT, Negative Control, Western Blot, Expressing, Quantitative RT-PCR, Plasmid Preparation