Tripz shrna plasmids

Manufactured by Horizon Discovery

TRIPZ shRNA plasmids are genetic constructs designed for the expression of short hairpin RNA (shRNA) sequences. These plasmids provide a platform for the stable, inducible knockdown of target genes in mammalian cell lines.

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Lab products found in correlation

Lenticrisprv2 puro, by Addgene (2 mentions) Prl sv40, by Addgene (2 mentions) Pgl3 ap 1, by Addgene (2 mentions) Pspax2, by Addgene (2 mentions) Luciferase assay buffer, by Promega (1 mentions) Dmem powder, by Merck Group (1 mentions) Phosphate buffered saline (pbs), by HiMedia (1 mentions) H3r17me2a, by Merck Group (1 mentions) Tritiated sam, by PerkinElmer (1 mentions) Lipofectamine, by Thermo Fisher Scientific (1 mentions) Trizol reagent, by Thermo Fisher Scientific (1 mentions) Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions) Reporter lysis buffer, by Promega (1 mentions)

3 protocols using tripz shrna plasmids

CARM1 and YY1 Regulation Assay

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TC100 insect medium (Ref: IML007), PBS (Ref: TL1006), Trypsin-EDTA solution 10× (Ref: TCL070) and Antibiotic antimyotic solution 100× (Ref: A002A) were obtained from HIMEDIA. The lipofectamine used for transfection was obtained from Invitrogen (Ref: 11668019). DMEM powder (Ref: D1152), MTT (Ref: M5655) and SAM (Ref: A7007) were obtained from Sigma-Aldrich (St. Louis, Missouri, USA). TRIzol reagent (Ref: 15596018) was obtained from ambion life technologies. The TRIPZ-shRNA plasmids used for stable cell line generation were obtained from Dharmacon (Ref: CARM1 sh -V3THS_319980 and YY1 sh-V2THS_219592). The commercial antibodies used in the study include Tubulin from calbiochem (Ref: CP06), CARM1 from abcam (Ref: Ab84370), YY1 from abcam (Ref: Ab12132) and H3R17me2a from Millipore (07–214). Tritiated SAM was obtained from Perkin-Elmer (Ref: NET155250UC). Luciferase assay buffer (Ref: E151A) and reporter lysis buffer (Ref: E397A) were purchased from Promega (Madison, Wisconsin, USA). CARM1 inhibitor (PRMT inhibitor V) was purchased from Calbiochem-Millipore (Ref: 217531).

Behera A.K., Kumar M., Shanmugam M.K., Bhattacharya A., Rao V.J., Bhat A., Vasudevan M., Gopinath K.S., Mohiyuddin A., Chatterjee A., Sethi G, & Kundu T.K. (2019). Functional interplay between YY1 and CARM1 promotes oral carcinogenesis. Oncotarget, 10(38), 3709-3724.

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Zebrafish Genetic Manipulation Protocol

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An sgRNA plasmid (MITF GFP 2X-sgRNA), which harbors a mitfa:GFP insert as well as a 2X sgRNA cassette (Fig. 2A), was generated for the zebrafish screen. The 2X sgRNA cassette from MAZERATI 2X (MAS2X) sgRNA (RRID Addgene_118844), also called MiniCoopR 2X sgRNA (gift from Dr. Len Zon), was amplified and introduced into mitfa:GFP plasmid using In-fusion cloning. Site-directed mutagenesis was performed to introduce NheI sites spanning sgRNA2 of the plasmid. MAS2X sgpten was cloned from MAS2X sgRNA (Supplementary Fig. S2A; ref. 44, 109 (link)). The U6-sgRNA plasmid (RRID Addgene_64245) was used along with mitfa:Cas9 plasmid and MiniCoopR:tdTomato (previously developed in the lab). LentiCRISPRV2 Puro (RRID Addgene_52961), psPAX2 (RRID Addgene_12260), and MD2 (RRID Addgene_12259) plasmids were obtained from Addgene. TRIPZ shRNA plasmids were purchased from Dharmacon expressing a nontargeting (NT) shRNA or shRNAs against human GRAMD1B. Clone IDs and hairpin sequences are in Supplementary Table S3. Control luciferase plasmid (pGL3, RRID Addgene_48743) and Renilla luciferase plasmid (pRL-SV40, RRID Addgene_27163) were obtained from Promega (E1751 and E2231) and pGL3-AP-1 was from Addgene (RRID Addgene_40342). Details of the oligonucleotide sequences used for cloning and sequencing are in Supplementary Table S3.

Suresh S., Rabbie R., Garg M., Lumaquin D., Huang T.H., Montal E., Ma Y., Cruz N.M., Tang X., Nsengimana J., Newton-Bishop J., Hunter M.V., Zhu Y., Chen K., de Stanchina E., Adams D.J, & White R.M. (2022). Identifying the Transcriptional Drivers of Metastasis Embedded within Localized Melanoma. Cancer Discovery, 13(1), 194-215.

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Generation of a MITF-GFP Zebrafish Screening Plasmid

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A sgRNA plasmid (MITF GFP 2X-sgRNA) which harbors a mitfa:GFP insert as well as a 2X sgRNA cassette (Fig.2A) was generated for the zebrafish screen. The 2X sgRNA cassette from MAZERATI 2X (MAS2X) sgRNA (RRID Addgene_118844), also called MiniCoopR 2X sgRNA, gift from Dr. Len Zon) was amplified and introduced into mitfa: GFP plasmid using In-fusion cloning. Site directed mutagenesis was performed to introduce NheI sites spanning sgRNA2 of the plasmid. MAS2X sgpten was cloned from MAS2X sgRNA (Supp. Fig. 2A) (44 (link),109 (link)). The U6-sgRNA plasmid (RRID Addgene_64245) was used along with mitfa:Cas9 plasmid and MiniCoopR:tdTomato (previously developed in the lab). LentiCRISPRV2 Puro (RRID Addgene_52961), psPAX2 (RRID Addgene_12260) and MD2 (RRID Addgene_12259) plasmids were obtained from Addgene. TRIPZ shRNA plasmids were purchased from Dharmacon expressing a non-targeting shRNA or shRNAs against human GRAMD1B. cloneIDs and hairpin sequences are in Supp. Table. S3. Control luciferase plasmid (pGL3 RRID Addgene_48743 ) and Renilla luciferase plasmid (pRL-SV40, RRID Addgene_27163) was obtained from Promega (E1751 and E2231) and pGL3-AP-1 was from Addgene (RRID Addgene_40342). Details of the oligonucleotide sequences used for cloning and sequencing are in Supp. Table. S3.

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