... 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...

...FHF2 Human Mouse IgG2a 114527 Anti-JIP-2/IB-2 [N135/37.2R] JIP-2/IB-2 Mouse Mouse IgG2a 114529 Anti-Pan-SAPAP...Anti-CDY1/2/L1/L2 [N143/36R] CDY1/2/L1/L2 Human Mouse IgG2a 206535 Anti-CDY1/2 [N143/38R] CDY1/2 Human Mouse...206763 Neuroligin-2 scFv [L107/90] L107/90 scFv Neuroligin-2 Mouse Mouse 206764 Neuroligin-2 scFv [L107/95...receptor Rat Mouse IgG2a 114484 Anti-Stonin-2 [N346/9R] Stonin-2 Human Mouse IgG2a 114485 Anti-NSD3 [N348...Pan-FHF-A Human Mouse IgG2a 114510 Anti-REEP1/2 [N326D/29R] REEP1/2 Mouse Mouse IgG2a 114511 Anti-Kv3.1b K+ ...Mouse Mouse IgG2a 114547 Anti-Mitofusin-2 [N171/17.4R] Mitofusin-2 Mouse Mouse IgG2a 114548 Anti-Mortalin...Mouse IgG2a 140065 Anti-SCG10/Stathmin-2 [L5/1R] SCG10/Stathmin-2 Rat Mouse IgG2a 140066 Anti-Co-Rest/RCOR1...

...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...

...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...

...48696 pANAP AnapRS E. coli 3-(6-acetylnaphthalen-2-ylamino)-2-amino-propanoic acid (Anap) Mammalian TAG Peter...Mammalian TAG Huiwang Ai 73544 pEvol-pAcFRS.2.t1 pAcFRS.2.t1 E. coli p-acetyl-l-phenylalanine (pAcF) Bacterial...Bacterial TAG Farren Isaacs 73546 pEvol-pAzFRS.2.t1 pAzFRS.2.t1 E. coli p-azido-l-phenylalanine (pAzF) Bacterial..._AnapRS AnapRS E. coli 3-(6-acetylnaphthalen-2-ylamino)-2-amino-propanoic acid (Anap) Mammalian TAG Simon...Jesse Rinehart 71403 pCMV-DnpK PylRS M. barkeri N6‐(2‐(2,4‐dinitrophenyl)acetyl)lysine (DnpK) Bacterial,...pDule-IBBN (G2) IBBN (G2) synthetase M. jannaschii 4-(2′-bromoisobutyramido)-phenylalanine (IBBN) and structurally...pDule2-IBBN (G2) IBBN (G2) synthetase M. jannaschii 4-(2′-bromoisobutyramido)-phenylalanine (IBBN) and structurally...

... Direct transfection into cells is not recommended. Can I order more than 2 µg of cloning grade DNA? At...feedback on this service. DNA Service Details Amount 2 µg Volume 20 µl Concentration 100 ng/µl (± 5ng/µl)... At this time, to order more than 2 µg of a single plasmid, multiple orders would need to be placed through...Can I directly transfect cloning grade DNA into cells? Cloning grade DNA is designed for use in cloning...

...Disable the other repair mechanisms Mammalian cells have 2 primary repair mechanisms besides HR - Non-homologous... However, if you have a larger knock-in (200 bp – 2 kb), a dsDNA donor will need to be used due to synthesis...accidentally change your protein of interest.   Fig. 2: Stop re-cutting with PAM and guide RNA disrupting.... Com., 8(2024), (2017). 10.1038/s41467-017-01836-2 Charpentier, M., Khedher, A. H. Y., Menerot, S., Brion...varying across cell types. Variance can be caused by cell cycle rates; rapidly diving cells will have higher... of your cells. Enrich S-phase cells HR operates predominantly in S and G2 phase of the cell cycle, when...HDR events, ensure cells are actively cycling through the cell cycle stages. If cells are overly confluent...

...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...

...non-specific proteins, and then elute YFP.  Option #2: Opto-Nanobodies (OptoNBs) purification You skip adding...blue lights. Which option do you choose?  Option #2 may sound like fiction, but light-controlled protein...experiments.  AsLOV2 Inertion Site mCherry #1 mCherry #2 GFP Loop 1 dark-induced dark-induced dark-induced...OptoNBs to control cell signaling The second application for OptoNBs takes us into cells: light-based control... regulation of endogenous signaling activity in cells.  Let’s shed some light on the key components of...their use for protein purification and controlling cell signaling. Key components of OptoNBs OptoNBs have...can bind, it co-localizes with its target at the cell membrane and when the OptoNB can’t bind, it localizes...

...3319, 3180 cm 2 Cellstack 2, Corning 3269, 1272 cm 2 Heat-inactivated FBS (HI-FBS) Pro-Tip Different brands... 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...

... post treatment (see figure 2). Induced cell death was confirmed by TUNEL staining.  The video below shows...fluorogen activating protein - dL5** in this case), and 2) a fluorogen (the TAP - targeted and activated photosensitizer...PMID: 26808669. PubMed Central PMCID: PMC4916159. 2. Jacobson, K., Rajfur, Z., Vitriol, E., & Hahn, K....HEK293 cell culture and in live zebrafish. For cell culture experiments, only dL5**-expressing cells treated...inactivate target proteins or cells while having little effect on neighboring cells. The photosensitizer used...Here Light-induced cell ablation with MG-2I-dL5** He et al tested out light-induced cell ablation with MG...effective killing of dL5** cells without death of neighboring bystander wild-type cells.   Zebrafish experiments...

...Induction of Human Germ Cell Fate from Pluripotent Stem Cells. Cell Stem Cell, 2015. 17(2): p. 178-94. PubMed... of human induced pluripotent stem cells. Stem Cell Rev, 2015. 11(2): p. 242-53. PubMed PMID: 25578634...differentiated somatic cell to revert back to a pluripotent stage (iPSC formation) [2, 3]. The iPSCs then...somatic cells via various methods. Some of the more common delivery methods include retrovirus [2], lentivirus...PubMed PMID: 16810240. 2. Takahashi, K., et al., Induction of pluripotent stem cells from adult human fibroblasts...three routes to regeneration. Nat Rev Mol Cell Biol, 2011. 12(2): p. 79-89. PubMed PMID: 21252997. 37. Merrell...differentiation of human ES cells and iPS cells into mature pancreatic insulin-producing cells. Cell Res, 2009. 19(...

...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...

...postmitotic, long-lived cell types. AAVs in the clinic Thus far, AAV vectors AAV1, AAV-2, AAV1-AAV2 hybrids...neutralizing antibodies against some AAV types, AAV1, 2, 3 and 5 are thought to be as high as 70% among the... 10.24 (2003): 1999-2004. PubMed PMID: 14566358.  2. Hirsch, M. L., et al. "Self-complementary AAV mediates... do so, their T-cells were grown in vitro, their mutations corrected, and the T-cells were transferred...functioning T cell repertoire (1). In this example, it was possible to transduce the T-cells outside the...a wide range of cell types including but not limited to muscle, liver, and brain cells. There are a multitude...chain (c) and is characterized by a lack of immune cell development and function. To date, gene therapy ...

...splitting transfected cells into 3 plates at different densities for selection). A 2% agarose solution is...transition from a 48-well cell plate to a 96-well assay plate. We generally freeze at least 2 ampules each of 4...of agarose, which is then overlaid with the firmer 2% agarose gel solution discussed above for picking.... a wide variety of cell lines, from HEK to CHO cells to neural and endocrine cells such as AtT-20, PC12...then handled. Additionally, many cell lines, especially the endocrine cell lines we most commonly work with...specific cell line, and its transfection efficiency. We often trypsinize the transfected cells on the day...increased). For some cells, add a drop of serum to aid in outgrowth. Depending on the cell type, we sometimes...

...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 ...