...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...

...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...

... 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...

...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...

...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...

...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...

... 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(...

...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...

...deletions in human cells using zinc finger nucleases. Genome Res. 2010 Jan;20(1):81-89. PubMed. 2. Bauer DE, ...with TALENs, in collaboration with the Porteus lab [2]. With the advent of CRISPR/Cas9, we began to explore...CRISPR plasmids into MEL cells by electroporation, sort your transfected cells via FACS, primer validation...article "Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9" from the Journal of Visualized...CRISPR/Cas9 to create genomic deletions in mammalian cell lines. Below Bauer and Canver discuss the motivations...deletions is to produce monoallelic deletion clones. Cells with just a single intact copy at a given locus ...screening by conventional PCR. Biallelically disrupted cells can be easily identified. Even in the case of clones...

... genetically encoded H2O2 probe is pQE-30-HyPer3 (2) which he will co-express together with a plasmid ...Pharmacology 2017 Jun 15;134:1-17. PubMed PMID: 27984002. 2. Bilan, Dmitry S. et al. “HyPer-3: A Genetically Encoded... Accelerating science and supporting scientists globally are the shared mission of both Seeding Labs ...

... Podcast Interview with Stem Cell Researcher Darrell Kotton 2. Stem cell scientists care about patients...explained that it took 50 people nearly 2 years to create the first stem cell line that could be used in clinical...prevent stem cell lines from becoming the new HeLa cells?), genome engineering and stem cells, and the difficulty...opportunity to see Shinya Yamanaka celebrate the tenth anniversary of his landmark Cell paper showing that it was...generate induced pluripotent stem cells (iPSCs) capable of forming nearly all cell types in the body from otherwise... happening. Stem cells scientists have certainly not lost site of that. 3. Stem cell science is hard, ...otherwise terminally differentiated cells simply by expressing four proteins Oct4, Sox2, Klf4, and cMyc...

...the specimen.  2. Fluorescence Activated Cell Sorter (FACs) A machine that sorts cells according to whether...photostability of your fluorescent protein when using 2-photon microscopy as this hasn't been well reported...PMID: 18703742. PubMed Central PMCID: PMC2606057. 2. Chtanova, Tatyana, et al. "Dynamics of neutrophil...labeling T cells, B cells, and antigen presenting cells, scientists have been able to decypher cellular dynamics...tracked by cell sorter giving immunologists the ability to count cells and statistically analyze cell movements...fast T cells could jump from one antigen presenting cell to another. Indeed, we now know that T cells are...2016)? How do dendritic cells migrate to lymph nodes to activate T cells (Celli et al 2008, Kitano et al...

...NGG-3′ (“N” can be any nucleotide base, followed by 2 guanines). This limits the use of ABEs as DNA sites... in the promoter region of gamma hemoglobin (HGB1/2) leads to continuous expression of fetal hemoglobin...primary human cells  Cell therapy approaches can use base editors to correct mutations in stem cells and return...primary human cell editing, the researchers tested ABE8 in CD34+ hematopoietic stem cells. ABE8 efficiently... In addition to stem cells the authors targeted 6 genes in primary human T cells using 8 selected ABE8s...to edit primary human T cells efficiently makes it a promising tool for T cell therapy development.  Reducing...be introduced into cells as a plasmid or as mRNA constructs. In primary human cells mRNA delivery of ABEs...

...PMC5118044. 2. Kim, Y., et al., The Generation of Human Induced Pluripotent Stem Cells from Blood Cells: An Efficient...blood are reprogrammed immediately after collection [2]. PBMCs display unique epigenetic signatures that ...human-induced pluripotent stem cells from cord blood and peripheral blood cells. Stem Cells, 2013. 31(3): p. 458...possible to reprogram differentiated cells into induced pluripotent stem cells (iPSCs) by the addition of reprogramming... somatic cells that are otherwise difficult to obtain. These can then be used to perform cell therapy ...in vitro. Find stem cell plasmids at Addgene Many sources of adult somatic cells have been probed for...Using Serial Plating of Reprogrammed Cells by Centrifugation. Stem Cells Int, 2016. 2016: p. 1329459. PubMed...