...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...

...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...

...status of MEK1/2 was tracked at 5 minute intervals via Western blot. Phosphorylation of MEK1/2 peaks at just...PubMed PMID: 28232577. Figures 2-4 were modified from this reference. 2. Regot, Sergi, Jacob J. Hughey...Treatment of the cells with a PP1/PP2A inhibitor leads to maintenance of phosphorylated MEK1/2 levels, suggesting...precisely control the timing of kinase activity. 2. Studying negative feedback loops of signaling pathways... studied the negative feedback on endogenous MEK1/2 phosphorylation by creating a short pulse of psRaf1...activity. They did this by exposing psRaf1 to a short 2 minute pulse of 500 nm light (turn on psRaf1) followed...suggesting PP1/PP2A is responsible for this MEK1/2 negative feedback. 3. Studying signaling pathways in...

...Unc5b Unc5b 28215 SI2-Unc5b Unc5b 28216 SI4-Jamc.2 Jamc.2 28217 SI3-Flrt3 Flrt3 36115 SI5-IGSF21a IGSF21a...plasmids that encode monoclonal antibodies to zebrafish cell surface receptors and secreted proteins. Plasmid...plasmids that encode monoclonal antibodies to zebrafish cell surface receptors and secreted proteins. The plasmids...antibodies are produced by transfecting mammalian cell lines and collecting the secreted monoclonal antibody...

...difficult to ignore [1, 2]. 2. Fusion to an obligate dimer or oligomer Several cell proteins normally self-associate.... J Cell Biol, 2003. 163(2): p. 257-69. PubMed PMID: 14581454. PubMed Central PMCID: PMC2173526. 2. Zacharias...Kd is unlikely to form dimers in a cell. The Kd of EGFP is 0.11 mM [2]. Following the simple logic outlined... nature are prone to dimerization (i.e. EGFP) [1, 2] or even form obligate tetramers (i.e. DsRed) [3]....organized smooth endoplasmic reticulum (OSER) whorls. 2)A dimerizing protein of interest fused to a dimerizing... designed to detect resulting in false positives [2, 4]. Solutions to the polymer problem There are some...Citrine) can be monomerized with an A206K mutation [2]. Addgene has many of these constructs and they are...

...same site, is 2’-fluoro (2’-F), which substitutes a fluorine for the hydroxyl group at the 2’ position. ...most common of these is 2’-O-methylation, where a methyl group is added to the 2’ hydroxyl group of the...them together, for example phosphorothioates with 2’-O-methyl modifications, proved more stability than...generated by introduction of a single photocleavable 2-nitrobenzyl linker in the 20-bp targeting region of...photocleavable gRNAs. Molecular Cell 81, 7, 1553-1565 (2021). https://doi.org/10.1016/j.molcel.2021.02.007 Moroz-Omori...evolved ways to prevent degradation of important cellular RNA molecules with post translational modifications...slightly more efficient and less toxic to mammalian cells (Allen, et al., Frontiers in Genome Editing).  Some...

...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...

...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...

...3319, 3180 cm 2 Cellstack 2, Corning 3269, 1272 cm 2 Heat-inactivated FBS (HI-FBS) *Pro-Tip* Different... 4 °C. Cell Lysis Buffer : 50 mM Tris HCl, 150 mM NaCl, 2 mM MgCl 2 Add the following to the 2 L sterile... solution can be stored at 4 °C for up to 2 months. After 2 months, discard the tube and thaw a new working... mL of PBS. Aspirate PBS and add 2 mL of 0.05% Trypsin/EDTA. Wait ~2 min. Neutralize trypsin by adding...Seed cells in CS2 Day 2: Seed cells in CS5 Day 3 (am): Transfect cells Day 7 (am): Harvest cells Equipment...times to obtain a single cell suspension (no clumps of cells). Pool cells from 2 x T-175 flasks. Adjust ...touching the cells when replacing media. T-175 flask, Corning 430825, 175 cm 2 Cellstack 5, Corning 3319...

...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 ...

... 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%)...

...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...

... Regenerative Medicine. Acta Naturae, 2010. 2(2): p. 18-28. PubMed PMID: 22649638. PubMed Central PMCID...specialized cell types [2]. We general speak of 4 levels of stem cell potency: Totipotency: When a cell has ...embryonic stem cells (ESCs), which I discuss in detail below [2]. Multipotency: When a cell can differentiate...mammalian development, cells found in the zygote through to the morula stage are totipotent [2-4]. Pluripotency...PubMed Central PMCID: PMC4686367. 2. Jaenisch, R. and R. Young, Stem cells, the molecular circuitry of pluripotency...Pharmacological Sciences, a Cell Press reviews journal. Stem cells are special types of cells that can develop ...pluripotent cells can be thought of as descendants of totipotent cells. An example of pluripotent cells is embryonic...

...into account that HDR is rare, usually 2-5% of the edited cells in our hands, these combined pitfalls ...hundred clones to find the right one Step 2 – Edit the cells a second time using a repair template that...improving editing accuracy by preventing re-editing and (2) optimizing “cut-to-mutation distance”. Furthermore...Additional CRISPR Resources on Addgene's CRISPR Guide Page 2. Optimizing "cut-to-mutation distance"  Even though... wisely (and let them cut close).   Figure 2: Relationship between cut site to mutation distance...PMID: 23287718. PubMed Central PMCID: PMC3795411. 2. Mali, Prashant, et al. "RNA-guided human genome engineering...sequence change the cell must undergo homology-directed repair (HDR), a distinct cellular DNA repair pathway...

...ImageJ plugins JACoP and Coloc 2, BioImageXD, and customized CellProfiler pipelines. Image analysis software...optimized for diverse cellular environments. Since colocalization relies upon the detection of 2 independent fluorescent...Markers of Subcellular Localization Poster here! References 1. Allen Institute Plasmid Pages 2. Cooper ... to the lysosome where they are finally degraded (2, 3). Once you've created your fusion protein, it's...colocalization analysis example is given in Figure 2 (adapted from 4) - Dunn et al. 2011 provides an in-depth...organs, cells comprise compartments and structures, called organelles. Take a sneak peak inside a cell with...vesicles via the secretory pathway to their subcellular or extracellular destination. At the end of their life...

...the start codon of retinal cell-type specific genes from mice. Strategy #2 (ProB): Create promoters using...horizontal cells transduced by both AAVs would express Cre-mCherry and GFP.   Figure 2: Synthetic...doi.org/10.1038/s41593-019-0431-2  Neff, E.P. An AAV library for retinal cell expression in mouse, macaque... horizontal cells, and Cre-dependent-GFP expresses in horizontal cells and ganglion cells, only the horizontal... in retinal cells. To do this, the group developed a new strategy for finding retinal cell-type specific...and high cell-type specificity.  Strategy #3 (ProC): Create promoters by repeating mouse cell type specific... to test for cell type-specific expression of GFP. Half (113) expressed in retinal cells four weeks post-injection...

... 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...

...can browse entries by cell line groups, such as their vaccine production, SARS-CoV-2 research, or adenovirus...Knowledge Resource. J Biomol Tech. 2018; 29(2): 25-38....in which the cell line was described. Using Cellosaurus Cellosaurus is searchable by cell line name (recommended...resource called Cellosaurus, a growing cell line information database describing nearly 145,000 cell lines. Cellosaurus... Cellosaurus Cellosaurus seeks to identify and describe all cell lines used in research, using four methods... related cell lines when annotating a cell line Community submissions via email: cellosaurus@sib.swiss...with cell line catalogs and collections. Importantly, Cellosaurus also identifies problematic cell lines...

...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...