Addgene inc lenticrispr v2 plasmid 52961 Lenticrispr V2 Plasmid 52961, supplied by Addgene inc, used in various techniques. hope that helps, - Neville. Packaging plasmid(s)LVGag, Pol, RevTat 3.Envelope plasmidLVVSV-G LV12323 Delivery. Plasmid lentiGuide-Puro from Dr. Feng Zhang's lab contains the inserts S. pyogenes sgRNA cassette and Puromycin resistance and is published in Nat Methods. Lentiviral titer was determined using HeLa FlpIn TRex cells, plated at 70% confluence. Cloning and expression of guide RNA T7 in vitro expression from a plasmid To produce guide RNA by in vitro transcription with T7 RNA polymerase, the guide RNA sequence can be cloned into a variety of plasmids (see AddGene website). Packaging plasmid(s)LVGag, Pol, RevTat 3.Envelope plasmidLVVSV-G LV12323 Affiliations. The editing strategy and design described above can be used to create similar donor plasmids for introducing tags into your gene of interest. Media.addgene.org created by Benjie Chen. AMPure XP-PCR purification (recommended) 1. This plasmid is available through Addgene. Corrector drugs reactivating mutant p53 are of high clinical value for cancer therapy. identify small drug-like compounds that restore wild-type activity to mutant forms of p53 and exhibit anti-tumor effects. Single-lysosome mass spectrometry (SLMS) integrates lysosomal patch-clamp recording and induced nanoESI/MS for concurrent metabolic and electrophysiological profiling of individual enlarged lysosomes. A brief and partial description of how to use this virus: Start by optimizing the infection conditions in your cell line in order to achieve 30-50% infection efficiency, corresponding to a multiplicity of infection (MOI) of ~0.51. sgAF10-1 and 2 plasmids were gifts of Or Gozani (Stanford University). Brunello library: Addgene 73178 (in lentiGuide-Puro) and Addgene 73179 (in lentiCRISPRv2) Calabrese library: Addgene 92379 (Set A), 92380 (Set B) Dolcetto library: Addgene 92385 (Set A), 92386 (Set B) Plasmid lentiCRISPRv2 puro from Dr. Brett Stringer's lab contains the insert P2A-puro and is published in Sci Rep. 2019 Mar 20;9(1):4902. doi: 10.1038/s41598-019-41277-z. lentiCRISPRV2 2. After you have inserted your gRNA, you should use hU6-F (5'-GAGGGCCTATTTCCCATGATT-3') or LKO.1 5(5'- GACTATCATATGCTTACCGT-3') to sequence Rest of the gRNAs were designed and cloned into lentiCRISPRv2 (Addgene, catalog no. We further investigated whether bone marrow macrophage-expressed Metrn is sufficient to regulate HSPC proliferation and mobilization. Plasmid lentiCas9-Blast from Dr. Feng Zhang's lab contains the inserts Cas9 and Blasticidin resistance and is published in Nat Methods. doi: 10.1158/0008-5472.CAN-16-2159. Durairaj et al. 72hs later, infected cells were treated with 1 ug/ml puro (Gibco) for screening. 2. A brief and partial description of how to use this virus: Start by optimizing the infection conditions in your cell line in order to achieve 30-50% infection efficiency, corresponding to a multiplicity of infection (MOI) of ~0.51. Thus, monitoring and restoring functional PR expression is important in order to sensitize tumor cells to progesterone therapy in endometrial cancer. 1 Broad Institute of MIT and Harvard 7 Cambridge Center Cambridge, MA 02142, USA. Single-lysosome mass spectrometry (SLMS) integrates lysosomal patch-clamp recording and induced nanoESI/MS for concurrent metabolic and electrophysiological profiling of individual enlarged lysosomes. Brunello library: Addgene 73178 (in lentiGuide-Puro) and Addgene 73179 (in lentiCRISPRv2) Calabrese library: Addgene 92379 (Set A), 92380 (Set B) Dolcetto library: Addgene 92385 (Set A), 92386 (Set B) The resulting amplicons were PCR-purified (Qiagen), digested with Esp3I (Fisher Scientific) and cloned into either lentiGuide (pXPR_003, Addgene 52963) or lentiCRISPRv2 (pXPR_023, Addgene 52961). lentiCRISPRV2 2. 2014 Aug;11(8):783-4. doi: 10.1038/nmeth.3047. Ferroptosis, a non-apoptotic form of cell death marked by iron-dependent lipid peroxidation1, has a key role in organ injury, degenerative disease and vulnerability of therapy-resistant cancers2. For lentiviral transduction, HEK293T cells were transfected with pLX304 plasmids, FUGW, or LentiCRISPRv2 (Addgene) (together with VSVG and psPAX2 plasmids). Tumor xenograft Metrn cKO also increased the number of CFU-Cs in the blood and HSCs Just as we will need to deploy a range of tools to effectively treat various diseases, we will need multiple delivery modes to ensure molecular tools reach the correct tissues and cell types. Cloning and expression of guide RNA T7 in vitro expression from a plasmid To produce guide RNA by in vitro transcription with T7 RNA polymerase, the guide RNA sequence can be cloned into a variety of plasmids (see AddGene website). shRNA and sgRNA sequences are listed in Additional file 3: Table S2. 2. Guide RNAs (gRNAs) targeting the amino acid coding sequence of human ATF4 were cloned into the lentiCRISPRv2 lentiviral vector (#52961, Addgene) with selectable marker for puromycin. Current Global rank is 34,795, site estimated value 63,528$ #addgene #bioinformatics lab notebook #plasmid #what is a plasmid. gRNA pooled library in lentiCRISPRv2 1 $600 Add to Cart Pooled Library 1000000049 gRNA pooled library in lentiGuide-Puro + lentiCas9-Blast plasmid and include Addgene in the Materials and Methods of your future publications. A large fraction of human tumors inactivate the major tumor suppressor p53 by mutations. Expression of progesterone receptor (PR) is a favorable prognostic marker for multiple solid tumors. The numbers of LSK cells and HSCs were slightly decreased in the bone marrow but dramatically increased in the blood of Metrn f/f;LysM mice (Figures 2A2D ). gRNA pooled library in lentiCRISPRv2 1 $600 Add to Cart Pooled Library 1000000049 gRNA pooled library in lentiGuide-Puro + lentiCas9-Blast plasmid and include Addgene in the Materials and Methods of your future publications. MDA-MB-231 cells (1.4 10 8) were transduced with a pooled genome-wide lentiviral sgRNA library in a Cas9-containing vector (Addgene, 90294) at a multiplicity of infection of 0.3. Pool PCR products into an eppendorf (15-30 L per well is typically sufficient). Lentiviral particles were generated by transfection HEK293T cells with lentiCRISPRv2-gRNA construct psPAX2 and pMD2.G (Addgene) at a ratio of 4:3:1, respectively. This plasmid is an updated version of the original lentiCRISPR (Addgene plasmid #49535) IMPORTANT: The primers suggestions listed above are for gene inserts that exist in the untouched vector. Durairaj et al. A large fraction of human tumors inactivate the major tumor suppressor p53 by mutations. The resulting amplicons were PCR-purified (Qiagen), digested with Esp3I (Fisher Scientific) and cloned into either lentiGuide (pXPR_003, Addgene 52963) or lentiCRISPRv2 (pXPR_023, Addgene 52961). Briefly, the single guide RNA targeting FOXD1 was cloned into lentiCRISPRv2 (Addgene#52961) which contained two expression cassettes, hSpCas9 and the chimeric guide RNA. For the guide sequence TGCCCTTGCAGGTGAAGCTC, the following primers should be ordered for cloning into the BsaI-digested plasmid DR274 SnapGene Viewer is free software that allows molecular biologists to create, browse, and share richly annotated sequence files. Lentivirus was packaged, and cells were infected with the virus and selected with puromycin for 10 days to establish stable cell line. Plasmid lentiGuide-Puro from Dr. Feng Zhang's lab contains the inserts S. pyogenes sgRNA cassette and Puromycin resistance and is published in Nat Methods. Gain unparalleled visibility of your plasmids, DNA and protein sequences. Human Brunello CRISPR knock-out pooled library was a gift from David Root and John Doench (Addgene #73178). identify small drug-like compounds that restore wild-type activity to mutant forms of p53 and exhibit anti-tumor effects. A brief and partial description of how to use this virus: Start by optimizing the infection conditions in your cell line in order to achieve 30-50% infection efficiency, corresponding to a multiplicity of infection (MOI) of ~0.51. This plasmid is an updated version of the original lentiCRISPR (Addgene plasmid #49535) IMPORTANT: The primers suggestions listed above are for gene inserts that exist in the untouched vector. For the guide sequence CTCAACTCAGCCCTCAACAA, the following primers should be ordered for cloning into the BsaI-digested plasmid DR274 Cloning and expression of guide RNA T7 in vitro expression from a plasmid To produce guide RNA by in vitro transcription with T7 RNA polymerase, the guide RNA sequence can be cloned into a variety of plasmids (see AddGene website). 2014 Aug;11(8):783-4. doi: 10.1038/nmeth.3047. doi: 10.1158/0008-5472.CAN-16-2159. Packaging plasmid(s)LVGag, Pol, RevTat 3.Envelope plasmidLVVSV-G LV12323 This plasmid is available through Addgene. transfer plasmid (e.g.lentiCRISPRv2 or lentiGuide-Puro) packaging plasmids pVSVg (AddGene 8454) and psPAX2 (AddGene 12260) transfer plasmidpackaging plasmids HEK293(F)T . Then the plasmid was packaged into lentivirus and transduced to hMSCs. We developed stable PR reporter gene containing endometrial cancer cell All vectors were confirmed by Sanger sequencing. Metformin is the usual first-line drug of choice to reduce blood glucose levels in patients with type 2 diabetes mellitus. 2017 Feb 15. pii: canres.2159.2016. 2014 Aug;11(8):783-4. doi: 10.1038/nmeth.3047. Corrector drugs reactivating mutant p53 are of high clinical value for cancer therapy. Before using this virus, Addgene strongly recommends reading the original publication (Doench et al., 2016). This plasmid is available through Addgene. There is no Cas9, so the titer is quite high. Plasmid LentiCRISPRv2GFP from Dr. David Feldser's lab contains the insert GFP and is published in Cancer Res. Plasmid lentiCRISPRv2 puro from Dr. Brett Stringer's lab contains the insert P2A-puro and is published in Sci Rep. 2019 Mar 20;9(1):4902. doi: 10.1038/s41598-019-41277-z. Cloning and expression of guide RNA T7 in vitro expression from a plasmid To produce guide RNA by in vitro transcription with T7 RNA polymerase, the guide RNA sequence can be cloned into a variety of plasmids (see AddGene website). The numbers of LSK cells and HSCs were slightly decreased in the bone marrow but dramatically increased in the blood of Metrn f/f;LysM mice (Figures 2A2D ). After you have inserted your gRNA, you should use hU6-F (5'-GAGGGCCTATTTCCCATGATT-3') or LKO.1 5(5'- GACTATCATATGCTTACCGT-3') to sequence This plasmid is available through Addgene. For the guide sequence TGTTTCGTGAGGGGCGAGCT, the following primers should be ordered for cloning into the BsaI-digested plasmid DR274 Lentiviral titer was determined using HeLa FlpIn TRex cells, plated at 70% confluence. To allow constitutive expression of TP53 R249S mutant, the TP53 R249S sequence was cloned into pLV-EF1a-IRES-Hygro vector (Addgene plasmid no. The Cas9 in lentiCRISPRv2 was replaced with dCas9-KRAB (Addgene 46911) and the Puromycin resistance gene was replaced with Blasticidin resistance gene (Addgene 75112) for CRISPR-Cas9 knockdown. Packaging plasmid(s)LVGag, Pol, RevTat 3.Envelope plasmidLVVSV-G LV12323 Before using this virus, Addgene strongly recommends reading the original publication (Doench et al., 2016). AMPure XP-PCR purification (recommended) 1. 3 Department of Brain and Cognitive Sciences Massachusetts Institute of Technology Cambridge, MA 02139, USA. The Cas9 in lentiCRISPRv2 was replaced with dCas9-KRAB (Addgene 46911) and the Puromycin resistance gene was replaced with Blasticidin resistance gene (Addgene 75112) for CRISPR-Cas9 knockdown. Annotate features on your plasmids using the curated feature database. Plasmid LentiCRISPRv2GFP from Dr. David Feldser's lab contains the insert GFP and is published in Cancer Res. 85134). Before using this virus, Addgene strongly recommends reading the original publication (Doench et al., 2016). Briefly, two LRRK2 sgRNAs, GAGTCCAAGACGATCAACAG and AACGCTGGTCCAAATCCTGG, were inserted into LentiCRISPRv2 (Addgene). Delivery of molecular and cellular tools is perhaps the biggest barrier to widespread adoption of these treatments. Plasmid lentiCas9-Blast from Dr. Feng Zhang's lab contains the inserts Cas9 and Blasticidin resistance and is published in Nat Methods. Store, search, and share your sequences, files and maps. MDA-MB-231 cells (1.4 10 8) were transduced with a pooled genome-wide lentiviral sgRNA library in a Cas9-containing vector (Addgene, 90294) at a multiplicity of infection of 0.3. For the guide sequence TCAAGTAATCCGCAAACATG, the following primers should be ordered for cloning into the BsaI-digested plasmid DR274 Gene knockout was performed using the puromycin-resistant CRISPR-Cas9 lentiviral vector plentiCRISPR v2 (Addgene plasmid no. Viral supernatants were collected 4872hr following transfection and concentrated using the centrifugal filter (Merck Millipore) according to the manufacturer's protocol. A brief and partial description of how to use this virus: Start by optimizing the infection conditions in your cell line in order to achieve 30-50% infection efficiency, corresponding to a multiplicity of infection (MOI) of ~0.51. Metformin, the most prescribed antidiabetic medicine, has shown other benefits such as anti-ageing and anticancer effects14. This plasmid is available through Addgene. Distribute 100 L of pooled products to a 96-well round bottom plate 2ug envelop plasmid pMD2.G (Addgene Plasmid #12259) 8ug lentiCRISPR v2 (Addgene Plasmid #52961)) I use Genejuice as the transfection reagent. Distribute 100 L of pooled products to a 96-well round bottom plate transfer plasmid (e.g.lentiCRISPRv2 or lentiGuide-Puro) packaging plasmids pVSVg (AddGene 8454) and psPAX2 (AddGene 12260) transfer plasmidpackaging plasmids HEK293(F)T . Site is running on IP address 104.22.9.34, host name 104.22.9.34 ( United States) ping response time 20ms Good ping. By continuing to use this site, you agree to the use of cookies. ZERO BIAS - scores, article reviews, protocol conditions and more We further investigated whether bone marrow macrophage-expressed Metrn is sufficient to regulate HSPC proliferation and mobilization. This plasmid is available through Addgene. The titer difference is because lentiGuide-Puro contains only the sgRNA and puro resistance. Bioz Stars score: 99/100, based on 1 PubMed citations. This plasmid is available through Addgene. Pool PCR products into an eppendorf (15-30 L per well is typically sufficient). CMV-EGFP lentiviral transfer 2.9. This website uses cookies to ensure you get the best experience. 52691) vector as previously described . SnapGene Viewer. 2014 Aug;11(8):783-4. doi: 10.1038/nmeth.3047. Researchers have developed a variety of endogenous gene tagging systems. 2 McGovern Institute for Brain Research Massachusetts Institute of Technology Cambridge, MA 02139, USA. This plasmid is available through Addgene. 2017 Feb 15. pii: canres.2159.2016. SLC46A3-deactivated T47D cell lines were generated through infection with lentiCRISPRv2-Opti (Addgene 163126) vectors encoding Cas9 and single-guide RNAs (sgRNAs) . lentiCRISPRV2 2. 2.5. Human Brunello CRISPR knock-out pooled library was a gift from David Root and John Doench (Addgene #73178). Metrn cKO also increased the number of CFU-Cs in the blood and HSCs However, PR expression is reduced or lost in malignant tumors. Before using this virus, Addgene strongly recommends reading the original publication (Doench et al., 2016). We have a different vector (Cas9-Blast) that you can use to make a stable cell line with Cas9 and select for it using blasticidin resistance. CMV-EGFP lentiviral transfer Generation of single-cellderived p53 KO liver organoids was performed. Ferroptosis, a non-apoptotic form of cell death marked by iron-dependent lipid peroxidation1, has a key role in organ injury, degenerative disease and vulnerability of therapy-resistant cancers2. SLC46A3-deactivated T47D cell lines were generated through infection with lentiCRISPRv2-Opti (Addgene 163126) vectors encoding Cas9 and single-guide RNAs (sgRNAs) . lentiCRISPRV2 2. Monitoring and restoring functional PR expression is reduced or lost in malignant.. 2014 Aug ; 11 ( 8 ):783-4. doi: 10.1038/nmeth.3047 endometrial cancer cell < a href= '' https //www.bing.com/ck/a! U=A1Ahr0Chm6Ly9Szwdvlm5Vcnvzagnoyxjnzs5Jb20Vbwvkaweuywrkz2Vuzs5Vcmcv & ntb=1 '' > lentiGuide-Puro < /a > Delivery identify small drug-like compounds that restore wild-type to. 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