...guide RNA (gRNA) for biochemical purification (Fig. 2). For additional information and protocols, check ... web page . Figure 1: iChIP general scheme Figure 2: enChIP general scheme Fujii Lab Plasmids Individual... of interest 85586 gRNA Cloning Vector Bbs I ver. 2 An empty sgRNA expression vector. sgRNA sequence can... into BbsI site. gRNA scaffold has efficient ver. 2 structure. 92220 pLenti_dCas9-2xAM Lentiviral plasmid...Mohlin S. Biochem Biophys Res Commun. 2018 May 5;499(2):291-298. doi: 10.1016/j.bbrc.2018.03.150. PubMed ... mammalian cells 48880 3xFNLDD/pMXs-I2 iChIP plasmid, expresses 3xFNLDD in mammalian cells 48972 3xFNLDD...mammalian cells 49536 3xFNLDD/pMXs-puro iChIP plasmid, expresses 3xFNLDD in mammalian cells 51240 3xFLAG-dCas9...

...transiently transfected cells. Co-expression of both plasmids in HEK293 cells could cause up to 2~3-fold repress...could cause up to 300-fold repress on targeted genes. 2. Two-plasmid CRISPRi system for mammalian gene knockdown...promoter and sgRNA targeting GFP (NT1) 46917 pU6-sgCXCR4-2 Human pSico-based U6 vector containing murine U6 promoter... UAS promoter (negative control) 46918 pU6-sgCD71-2 Human pSico-based U6 vector containing murine U6 promoter...Weissman JS, Arkin AP, Lim WA. Cell . 2013 Feb 28;152(5):1173-83. doi: 10.1016/j.cell.2013.02.022. PubMed PMID...JA, Lim WA, Weissman JS, Qi LS. Cell . 2013 Jul 9. doi: 10.1016/j.cell.2013.06.044. PubMed PMID 23849981...expression of arbitrary genes in bacteria or human cells ( Qi et al. ). This CRISPR interfering system (CRISPRi...

...Feng G, Sharp PA, Zhang F. Cell . 2014 Oct 9;159(2):440-55. doi: 10.1016/j.cell.2014.09.014. Epub 2014 Sep... Aug 29. Erratum in: Cell. 2013 Oct 10;155(2):479-80. PubMed . Genome engineering using the CRISPR-Cas9...SpCas9n with 2a-Puro and 2a-EGFP are also available. 2. SpCas9 (or SpCas9n, D10A nickase) + CRISPR RNA array... system - lentiCRISPR - sgRNA and SpCas9 together 2 vector system - lentiCas9-Blast and lentiGuide-Puro...two MS2 RNA aptamers at the tetraloop and stemloop 2 The MS2-P65-HSF1 activation helper protein Full references...backbone with MS2 loops at tetraloop and stemloop 2 and EF1a-zeo resistance marker. Contains BsmBI sites...inverted terminal repeats (ITR) from AAV serotype 2. SaCas9 only: This plasmid ( PX600, #61592 ) contains...

...yields are generally lower, with a minimum titer of 2 × 10 12 GC/mL and a typical titer of 7 × 10 12 GC/... 10 weeks. This breaks down as follows: Request 1–2 days Look for the banner on an eligible plasmid page... your request. We will send you an email within 1-2 days with a response. Most requests will be approved...make sure it is ready for your experiments. Shipping 2–7 days We will email you with tracking information... tools that effectively transduce many mammalian cell types. AAV is used in a wide range of experiments...

...Laplante M, Sabatini DM. Cell. 2012 Apr 13;149(2):274-93. doi: 10.1016/j.cell.2012.03.017. PubMed PMID...known as RPS6KB1; Ribosomal protein S6 kinase B1 TSC1/2 TSC1 TSC2 Tuberous sclerosis VHL Von Hippel-Lindau...Rictor RPTOR independent companion of MTOR, complex 2 SGK Serum/glucocorticoid regulated kinase 1 Return... (mTOR) is a key metabolic regulator controlling cell growth and proliferation. mTOR, a serine/threonine...signals to determine the nutritional state of the cell and regulates the switch from anabolic to catabolic...growth factors, including insulin. mTORC2 influences cell survival and cytoskeletal organization. Cancer Impact... promotes the conditions needed for uncontrolled cell growth and proliferation, including an abundant ...

...46, JNK1, JNK1A2, JNK21B1/2, PRKM8, SAPK1, SAPK1c MAPK9 JNK-55, JNK2, JNK2A, JNK2ALPHA... PRKM11, SAPK2, SAPK2B, p38-2, p38Beta MAPK12 ERK3, ERK6, P38GAMMA, PRKM12, SAPK...Mitogen-activated protein kinases (MAPKs), also known as extracellular signal-regulated kinases (ERKs), are serine/...threonine-specific protein kinases. They regulate diverse cellular programs by acting as an integration point for...

... mutations in the gene methyl-CpG binding protein 2 ( MECP2 ). MECP2 Rett syndrome is an X-linked disorder...X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat Genet . 23, 185–188. (Link opens in a new window... Cuddapah et al. 2014. Methyl-CpG-binding protein 2 (MECP2) mutation type is associated with disease severity... Specific mutations in methyl-CpG-binding protein 2 confer different severity in Rett syndrome. Neurology...males with mutations in Methyl-CpG binding protein 2. Am J Med Genet B Neuropsychiatr Genet . 180, 55–67...Unpublished Joost Gribnau Human Cell Line Models Induced pluripotent stem cells (iPSCs) are a powerful system...opens in a new window) LUHMES cell line , an immortalized pre-neuronal cell line that can be differentiated...

...different inherited genes: Presenilin 1, Presenilin 2, and APP gene.The majority (>90%) of individuals develop...study aggregation. Saha et al. Nat Commun. 2023 Feb 2. Study the C terminal domain of TDP-43 to better understand... rodent species. Boender et al. Sci Adv. 2023 Jun 2. Use Cas9 in astrocytes. Endo et al Science. 2022 ...with the iPSC toolbox . Lam et al. bioRxiv. 2022 Dec 2. Use PiggyBac plasmids with tet-inducible expression...protein-protein interactions and nerve cell function within the cell. Parkinson's Disease Parkinson’s disease...inducible vector . Harley et al. Cell Rep. 2023 Dec 26. Induce CNS cells with concomitant expression of ...with the disease and implicate a wide range of cellular processes. The vast majority of cases (90-95%)...

...Layer 2-3_IT Isocortex 230803 pAAV-AiE2638m-minBG-iCre(R297T)-BGHpA AiP20142 AiE2638m Cre Layer 2-3_IT ...Layer 2-3_IT Isocortex 220727 pAAV-AiE2543m-minBG-iCre(R297T)-BGHpA AiP2048 AiE2543m Cre Layer 2-3_IT ...Layer 2-3_IT Isocortex 230402 pAAV-AiE0680m-minBG-iCre(R297T)-BGHpA AiP15072 AiE0680m Cre Layer 2-3_IT ...to target a variety of cell populations in different brain regions. Labeled cell populations were tested...to primate neocortex. Cell Rep. 2021 Mar 30;34(13):108754. doi: 10.1016/j.celrep.2021.108754. PMID: 33789096...regulatory sequences — termed enhancers — to drive cell type-specific expression. The Allen Institute for...differential chromatin accessibility across different cell types and cloned into an AAV backbone. Most enhancers...

... Morris CellTag Barcode Library Version 2 Ready-to-use lentiviral particles carrying version 2 of the ...the CellTag barcoding library. Version 2 of the Celltag library contains 4934 barcodes to combinatorially...carrying version 1 of the CellTag barcoding library. Version 1 of the Celltag library contains 19973 barcodes...barcodes to combinatorially index cells for single-cell analysis of clonal dynamics. This library expresses...combinatorially index cells for single-cell analysis of clonal dynamics. This library expresses EGFP for easy visualization...carrying version 3 of the CellTag barcoding library. Version 3 of the Celltag library contains 5737 barcodes...

...DSB and 2. a specific edit close to the gRNA PAM site. When a repair template is present, the cell may repair...Cas enzymes? CasPEDIA is an encyclopedia of Class 2 CRISPR systems with wiki entries describing enzyme...sequence. DSBs are preferentially repaired in the cell by non-homologous end joining (NHEJ), a mechanism...

...diphtheria toxin receptor fused to GFP for studying cell ablation. 2 Jessell , Azim 45580 pAAV-flex-taCasp3-TEVp...constitutive Expression of the tet-off transactivator (tTA) 2 Gradinaru 99120 pAAV-ihSyn1-tTA Inducible Synapsin...Scanziani Tools for Cell Ablation These AAV encode tools that can be used for cell ablation. ID Name Expression...Transactivators Affinity Purification Neurophysiology Cell Ablation Tetracycline Transactivators and Inducible...expression of designer pro-taCasp3 and TEVp for studying cell ablation. 5 Shah , Wells Don’t See What You’re Looking...

... a new vial of early passage cells. Approximately 2 weeks post-thaw, cell culture supernatant is tested...item (see maps section for images). Figure 2: AAV Pro cells were transduced with either pAAV-Ef1a-mCherry-IRES-Cre...suggests that titers obtained using ddPCR are generally 2–3 fold higher than those achieved using the standard... preparations are added to cells in culture. Three to five days later, cell cultures are inspected for...involves transducing cells with serial dilutions of the lentiviral vector, harvesting cells several days later... (55634-AAVrg) alone at 1.7E6 viral genomes (vg)/cell, pAAV-CAG-FLEX-rc [Jaws-KGC-GFP-ER2] (Addgene 84445...Production Lentiviral preparations are produced in 293T cells using the 2nd generation psPAX2 and pMD2.G packaging...

...par with ZF arrays, if not slightly lower. Figure 2: Simplified representation of the Voytas/Bogdanove...variety of backbones in just a few steps ( Figure 2 ). The Bogdanove group also hosts web-based software...mammalian transcription. Nat Biotechnol. 2011 Feb;29(2):149-53. PMID: 21248753 . TALEs for the masses. Rusk.... Targeted gene disruption in somatic zebrafish cells using engineered TALENs. Sander JD, Cade L, Khayter...

...RNA-guided regulation of transcription in eukaryotes. Cell . 154(2):442-51. PMID: 23849981 Ishino Y, Shinagawa ...Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell . 163(3):759-71. PMID: 26422227 Zetsche...transcribed to make the pre-CRISPR RNA (pre-crRNA). (2) The pre-crRNA is processed into individual crRNAs...2015. Class 1 (Multi-subunit effector complex) Class 2 (Single multi-domain effector) Type I (Cas3) Type ... with unprecedented speed and specificity. Figure 2: An overview of CRISPR and NHEJ/HDR. The Cas9/gRNA...genomic loci in living human cells by an optimized CRISPR/Cas system. Cell . 155(7):1479-91. PMID: 24360272... for Mapping Multiprotein Complexes in Human Cells. Cell Rep . 13(3):621-33. PMID: 26456817 Fu Y, Sander...

...degrades the mRNA. RISC includes argonaute protein 2 (Ago2), which facilitates cleavage of the target. ... International Journal of Biomedical Science, 13 (2), 48–57. PubMed (Link opens in a new window) . Moore... in therapeutic settings. As a tool in mammalian cell systems, silencing is achieved through the delivery...). Origins and mechanisms of miRNAs and siRNAs . Cell, 136 (4), 642–655. PubMed (Link opens in a new window...

...recombining shuttle plasmids Vogelstein 16401 pAdEasy-2 Adenoviral For recombining shuttle plasmids; for use...transgene under CMV promoter Vogelstein 16407 pAdEasy 2-GFP beta-gal Shuttle Test plasmid that contains β-...target a broad range of dividing and non-dividing cell types with almost 100% efficiency. Unlike lentiviruses...recombination of adenovirus plasmids in either mammalian cells or microorganisms, including bacteria and yeast....can then be transfected into mammalian packaging cell lines to generate adenovirus particles. Read our...

...phage display. J Immunol Methods. 2000 Aug 28;242(1-2):159-81. Carlos Barbas Vector system for expression...+J phagemid libraries. Immunol Lett. 2015 Oct;167(2):95-102. Joanna Bereta Sybody Generation Toolbox for...expressing these in bacteria, mammalian cells, yeast, plants, or insect cells. Benefits include consistency between... research purposes include animals or hybridoma cells, but are difficult to generate due to expense and...monoclonal antibodies (R-mAbs) expressed in mammalian cells. Monoclonal antibodies are derived from an individual...transiently transfected into mammalian tissue culture cells, the R-mAbs are secreted into the culture media ...

...gene modification. Maeder ML et al. Mol Cell. . 2008. Jul 25. 31(2):294-301. PubMed PMID 18657511 . Oligomerized...OPEN OPEN gGTCATCTGCcattcGAGGCGGACa REST corepressor 2 OZ553 and OZ554 OPEN OPEN gCTCACCATCtgttggaTGCGATGGAa...

...Regulation Ben Deverman 2:00 PM Cre-DOG and Combinatorial Strategies Connie Cepko 2:45 PM Break 3:00 PM (...GCaMP6f-P2A-nls-dTomato Detailed protocol: AAV Production in HEK293 Cells Please share materials by uploading to your Dropbox...