tre gfp blast  (Addgene inc)

Journal: JID Innovations

Article Title: A Series of BRAF- and NRAS-Driven Murine Melanoma Cell Lines with Inducible Gene Modulation Capabilities

doi: 10.1016/j.xjidi.2021.100076

Figure Lengend Snippet: Loss of Cre expression and rtTA3 activation by exogenous Cre. ( a ) Cre expression was tested by western blot and found to be absent in the 21 cell lines. Nevi isolated from hyperpigmented mouse skin of a BPP chimera was used as a positive control. ( b ) Parental M10M7 cells showed no rtTA3 activity likely owing to failed excision of the LSL cassette in the CAGS‒LSL‒rtTA3 allele. M10M7 cells were infected with lentivirus carrying TRE-GFP, followed by AdCre recombinase. On Dox treatment, cells infected with AdCre expressed GFP, indicating successful LSL removal and rtTA3 expression. Bar = 2 mm. AdCre, adenoviral Cre; BCC, Braf V600E ; Cdkn2a Δ/Δ ; BPP, Braf V600E ; Pten Δ/Δ ; Dox, doxycycline; NCC, Nras Q61R ; Cdkn2a Δ/Δ ; NPP, Nras Q61R ; Pten Δ/Δ .

Article Snippet: High-fidelity, codon-optimized Cas9 was cloned from pLenti-HF1RA-PGK-Puro , a gift from Lukas Dow (plasmid number 110860; Addgene, Watertown, MA), by replacing GFP in pLenti-TRE-GFP-Blast by In-Fusion cloning (catalog number 638911; Takara Bio).

Techniques: Expressing, Activation Assay, Western Blot, Isolation, Positive Control, Activity Assay, Infection

Journal: JID Innovations

Article Title: A Series of BRAF- and NRAS-Driven Murine Melanoma Cell Lines with Inducible Gene Modulation Capabilities

doi: 10.1016/j.xjidi.2021.100076

Figure Lengend Snippet: Inducible gene modulation in murine melanoma cell lines. ( a ) Schematic depicting the generation of stable cell lines expressing inducible Cas9 and sgRNA targeting GFP or Mafg . Mouse melanoma cell lines were infected with a lentiviral construct expressing Dox-inducible Cas9 and selected with blasticidin. Subsequently, TRE-Cas9‒harboring cells were infected with GFP-U6-sgGFP or GFP-U6-sgMafg1 and selected with hygromycin, and polyclonal populations were used for experiments. ( b ) TRE-Cas9‒harboring mouse melanoma cell lines were infected with a GFP-U6-sgGFP reporter construct. Western blot showed that Dox treatment resulted in Cas9 (Flag) expression and a decrease in GFP levels. ( c ) TRE-Cas9‒harboring M10M3 and M167M1 cells were infected with GFP-U6-sgGFP or GFP-U6-sgMafg1. Cells were then treated with Dox, and Flag-Cas9, GFP, and MAFG expressions were analyzed by western blot. Cas9 was expressed on Dox treatment, GFP was decreased in sgGFP-expressing cells, and MAFG was decreased in sgMafg1-expressing cells. ( d ) M10M3 and ( e ) M167M1 cells harboring TRE-Cas9 and sgGFP or sgMafg1 were plated at a low density, and colony-forming ability was examined, which is shown as percent surface area covered by colonies. ( f ) M10M3 and ( g ) M167M1 cells harboring TRE-Cas9 and sgGFP or sgMafg1 were plated in soft agar, and anchorage-independent growth was examined. Dox, doxycycline; sgGFP, sgRNA targeting GFP; sgMafg1, sgRNA targeting Mafg .

Article Snippet: High-fidelity, codon-optimized Cas9 was cloned from pLenti-HF1RA-PGK-Puro , a gift from Lukas Dow (plasmid number 110860; Addgene, Watertown, MA), by replacing GFP in pLenti-TRE-GFP-Blast by In-Fusion cloning (catalog number 638911; Takara Bio).

Techniques: Stable Transfection, Expressing, Infection, Construct, Western Blot

Journal: bioRxiv

Article Title: A versatile ES cell-based melanoma mouse modeling platform

doi: 10.1101/658260

Figure Lengend Snippet: Murine melanoma cell lines as a resource to complement chimera experiments in vitro and in vivo. (A) Melanoma cells from B TRE-shPten chimeras are maintained in Dox-containing media and restore Pten expression upon Dox removal. Western blot shows increased expression of Pten 3 and 6 days after Dox removal, while expression of GFP and AKT pS473 decrease. Total Akt and Hsp90 were used as loading controls. (B) Proliferation curve of B TRE-shPten melanoma cells under on-Dox and off-Dox conditions. (C) Focus formation of B TRE-shPten melanoma cells under on- Dox and off-Dox conditions. (D) Tumor growth of B TRE-shPten melanoma cells in Nu/Nu recipient mice. 5 mice each were kept on a regular diet (Off Dox, n=10 tumors), placed on a Dox diet two days prior to transplantation of tumor cells (On Dox, n=10 tumors), or switched from the On Dox condition to a regular diet 18 days after transplantation of tumor cells (On-Off Dox, n=10 tumors). (E) Survival of Nu/Nu mice shown in (E) bearing B TRE-shPten melanomas (n=5 for each cohort). (F) Western blot of BPP melanoma cells infected with pLenti-TRE-PtenWT, pLenti-TRE-PtenC124S, or pLenti-TRE-GFP lentiviruses. The effect of Dox-mediated induction of Pten expression on Akt phosphorylation (pS473 and pT308) is shown. BCC cells are used as a control for Pten expression, and total Akt and actin were used as loading controls. (G) Proliferation curve of BPP melanoma cells expressing TRE-PtenWT, TRE-PtenC124S, or TRE-GFP (H) Focus formation of BPP melanoma cells expressing TRE-PtenWT, TRE-PtenC124S, or TRE-GFP. (I) Tumor growth of BPP melanoma cells expressing TRE-PtenWT (n=10 tumors) or TRE-PtenC124S (n=10 tumors) transplanted in C57BL/6 mice. Recipient mice were placed on a Dox diet 2 days prior to melanoma cell transplantation. ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The Cre reporter was a gift from N. Geijsen (Addgene plasmid #62732). col1a1-EF1 -GFP was generated by removing TRE from col1a1-TRE and GFP from pLenti-GFP (Addgene plasmid #17448) and the EF1α core promoter from lentiCRISPRv2_puro (from T. Jacks) were inserted using standard cloning techniques. pLenti-TRE-GFP-Blast was generated by replacing the CMV promoter in pLenti-GFP-Blast with the TRE3G promoter from pRRL-TRE3G-GFP-PGK-Puro-IRES-rtTA3 (from J. Zuber). pLenti-TRE-PtenWT-Blast and pLenti-TRE-PtenC124S-Blast were created by replacing GFP in pLenti-TRE-GFP-Blast with mouse Pten wildtype or mutant cDNA.

Techniques: In Vitro, In Vivo, Expressing, Western Blot, Transplantation Assay, Infection

Journal: bioRxiv

Article Title: A versatile ES cell-based melanoma mouse modeling platform

doi: 10.1101/658260

Figure Lengend Snippet: Restoration of endogenous Pten expression in B TRE-shPten chimeras halts melanoma growth. (A) Fluorescent imaging of gross melanomas in B TRE-shPten chimeras where the inducible expression of shPten is linked to GFP. Taking chimeras off Dox for 14 days results in cessation of GFP expression. (B) Western blot showing expression of GFP, Pten, and phosphorylated (pS473 and pT308) and total Akt in melanomas from chimeras on Dox, or taken off Dox for 7 days. Actin was used as a loading control, and a tumor from a BCC chimera is shown for comparison. (C) Immunohistochemistry staining of Pten, GFP and Ki67 on tumors from B TRE-shPten chimeras on Dox or off Dox for 7 or 14 days. (D) Graph displaying the fold change in tumor volume over time in chimeras on Dox compared to chimeras that were taken off Dox on day 0. (E) Quantification of Ki67-positive nuclei per field in tumors from B TRE-shPten chimeras on Dox or off Dox for 7 or 14 days. * p < 0.05; ** p < 0.01; **** p < 0.0001.

Article Snippet: The Cre reporter was a gift from N. Geijsen (Addgene plasmid #62732). col1a1-EF1 -GFP was generated by removing TRE from col1a1-TRE and GFP from pLenti-GFP (Addgene plasmid #17448) and the EF1α core promoter from lentiCRISPRv2_puro (from T. Jacks) were inserted using standard cloning techniques. pLenti-TRE-GFP-Blast was generated by replacing the CMV promoter in pLenti-GFP-Blast with the TRE3G promoter from pRRL-TRE3G-GFP-PGK-Puro-IRES-rtTA3 (from J. Zuber). pLenti-TRE-PtenWT-Blast and pLenti-TRE-PtenC124S-Blast were created by replacing GFP in pLenti-TRE-GFP-Blast with mouse Pten wildtype or mutant cDNA.

Techniques: Expressing, Imaging, Western Blot, Immunohistochemistry, Staining

Journal: bioRxiv

Article Title: A versatile ES cell-based melanoma mouse modeling platform

doi: 10.1101/658260

Figure Lengend Snippet: Ectopic Pten re-expression impairs melanoma formation. (A) Kaplan-Meier analysis of tumor-free survival depicting the onset of melanoma formation in BPP TRE-Pten and BPP TRE-GFP chimeras. (B) Kaplan-Meier analysis showing the overall survival of BPP TRE-Pten and BPP TRE-GFP chimeras. (C) Quantification of tumor numbers in BPP TRE-Pten and BPP TRE-GFP chimeras. (D) Immunohistochemistry staining of Pten and GFP in BPP TRE-Pten and BPP TRE-GFP chimeras. (E) Quantification of melanomas with strong or weak/absent Pten staining intensity in melanomas from BPP TRE-Pten and BPP TRE-GFP chimeras. ns, not significant; * p < 0.05.

Article Snippet: The Cre reporter was a gift from N. Geijsen (Addgene plasmid #62732). col1a1-EF1 -GFP was generated by removing TRE from col1a1-TRE and GFP from pLenti-GFP (Addgene plasmid #17448) and the EF1α core promoter from lentiCRISPRv2_puro (from T. Jacks) were inserted using standard cloning techniques. pLenti-TRE-GFP-Blast was generated by replacing the CMV promoter in pLenti-GFP-Blast with the TRE3G promoter from pRRL-TRE3G-GFP-PGK-Puro-IRES-rtTA3 (from J. Zuber). pLenti-TRE-PtenWT-Blast and pLenti-TRE-PtenC124S-Blast were created by replacing GFP in pLenti-TRE-GFP-Blast with mouse Pten wildtype or mutant cDNA.

Techniques: Expressing, Immunohistochemistry, Staining