...interest, and stimulate or manipulate responses through neurological means like electric stimulation, drug administration...response to both of these stimulations. The best part? The chemical stimulation was sucrose…..sounds like...understand my obsession with the combination of chocolate and peanut butter), but it’s an undeniably difficult...them for downstream control – this is important later! Next, additional components are added - the ‘bells... the digestion of food, while brain tissue is relatively static). To operate both types of implanted ...microcontroller, NeuroStack coordinates optical stimulation of its associated fibers across multiple channels...it - the device has a rechargeable battery, is relatively small, and can be detached easily whenever necessary...

...humans. Then, one of them heard about our online platform, called Crowdfight, and filed a request for help...Crowdfight-COVID19 Crowdfight was born as a collaborative platform to help researchers working on COVID-19. Its origins...feeling helpless about the situation. But a few days later we had an idea. And this idea rapidly grew and became... became Crowdfight COVID-19, an online platform to connect researchers working on COVID-19 with scientists... designers and other professionals, joined the platform; while they are a minority, their help has been... to push certain projects. The idea behind the platform was simple: any person working on COVID-19 could...simpler and the volunteers helped with things like translations, bibliographic searches, etc.     Expanding...

...developing a moderate throughput functional genomic platform to annotate genetic perturbations that may play...attempting to characterize mutations in a pooled cell population, as mutations with strong phenotypic effects ...therapeutic interventions for cancer. This screening platform is an incredible boost to the cancer research ...

...tagless protein purification as well as reversibly regulation of endogenous signaling activity in cells.  Let...spatiotemporal resolution than pharmacological or genetic manipulation of the signaling pathway.  As a proof-of-principle...Ras and initiates the extracellular signaling-regulated kinase (ERK) signaling cascade. This signaling...results demonstrate that OptoNBs can be harnessed to regulate signaling pathways such as the Ras/Erk pathway...

...Anastasia continued profiling samples, and years later, several whole genome sequencing projects were launched...

...!   Epigenetics nation stuck with methylation correlation  In the 1980s, a lot of things were cool... slightly smaller population, something else was also suddenly cool: DNA methylation. In 1983, Andrew ...these studies report correlations without clearly testing whether a certain methylation state causes a condition...cause demethylation?  Dividing cells depend on DNA methyltransferase 1 (DNMT1) to copy methylation marks...site. So, when DNMT1 methylates the genome during DNA replication, it doesn’t methylate sites blocked by ...the original methylated strand is diluted to undetectable amounts: ergo, DNA demethylation! With optimization...allowing one to study the causal relationship between DNA demethylation and gene expression (Sapozhnikov...

... Congenital Disorders of Glycosylation (APCDG) and an international platform for CDG research, named the...NFIB- and NFIC-related human syndromes. As therapies are developed for specific NFI-related genetic disorders...on rare diseases through the BeHEARD Award. Congratulations to Paula Videira, Michael McMurray, and Richard...Addgene to advance their research on diseases of glycosylation, arginine mutagenesis, and neural development...development. Paula Videira: Congenital disorders of glycosylation Paula Videira is an Assistant Professor at the...CDG&Allies-PPAIN).  Congenital disorders of glycosylation (CDG) are a group of rare inherited metabolic...Immunological aspects of congenital disorders of glycosylation (CDG): a review. J. Inherit. Metab. Dis. 39,...

...solution later in this protocol. Isolating a Monoclonal Cell Population by Limiting Dilution Isolate cells...Protocols Cloning by Limiting Dilution Isolating a Monoclonal Cell Population by Limiting Dilution You may also...solution will be used to seed the 96-well plate. Sample Calculation First, use a hemocytometer to quantitate...cells/mL To seed one 96-well plate, make 10 mL of a 5 cell/mL solution. Calculate the total cells needed: ...into each well of a 96-well plate. By doing this, you are seeding the plate at an average density of 0.5... will help you focus the microscope later when scanning the plate for individual cells, which can be difficult...scan the plate for individual cells, which should be on the same focal plane. Scan the entire plate and make...

...KI-RAS, KRAS1, KRAS2, NS3, RASK2 LAT linker for activation of T cells LAT1, pp36 LCK lymphocyte-specific ...ADRM1 adhesion regulating molecule 1 ARM1, GP110, MGC29536, Rpn13 AP3B1 adaptor-related protein complex...polypeptide-related sequence A FLJ60820, MGC111087, PERB11.1 MICB MHC class I polypeptide-related sequence... IREL RFX5 regulatory factor X, 5 (influences HLA class II expression) - RFXANK regulatory factor X-associated...kinase, regulatory subunit 1 (alpha) GRB1, p85, p85-ALPHA PIK3R2 phosphoinositide-3-kinase, regulatory subunit...JULF2, KPG_004, NOP9 PF4 platelet factor 4 CXCL4, MGC138298, SCYB4 PF4V1 platelet factor 4 variant 1 CXCL4L1...

...detach cells. Plate 100 μl each into two separate 96-well flat-bottom plates. Keep one plate to allow for...“bulk”) that were not plated, freeze half of the cells for future plating. Plate the other half for screening...reaction (from 3.2.1 - 3.2.2 ). Plate onto a lysogeny broth (LB) agar plate with 100 μg/ml ampicillin and...CRISPR/Cas9 constructs. Plate sorted cells individually into 96-well round-bottom plates using sorter or by...dilution at 30 cells per 96-well round-bottom plate. Optimize plating the cell type used at limiting dilution...either grow as individual cells in 96-well flat bottom plate or in a 10 cm dish at low concentration so...derived clones can be picked and moved to a flat bottom 96-well plate. Allow the bulk cells to incubate at 37...

...efficiency of Cas9 cleavage is relatively high and the efficiency of HDR is relatively low, a large portion of...modifying the methylation state of cytosines in a gene’s promoter or by inducing histone acetylation or demethylation... Select Your Desired Genetic Manipulation Different genetic manipulations require different CRISPR components...Select gene and genetic element to be manipulated In order to manipulate a given gene using CRISPR, you will...Plan Your Experiment Overview Select Genetic Manipulation Select Expression System Design gRNA Know your...has important practical implications, because a population of cells expressing Cas9 and a gRNA will result... to utilize HDR for gene editing, a DNA repair template containing the desired sequence must be delivered...

...Reagents Protein phosphorylation is one of the most abundant forms of posttranslational modifications in...questions surrounding serine phosphorylation by adding this phosphorylated amino acid to the genetic code...improving the orthogonal translation system to make singly or multiply phosphorylated proteins (Pirman et ... serine phosphorylation ( Rinehart Human Serine Phosphopeptide Library ). The phosphorylation sites (“... ) and an optimized phosphoserine orthogonal translation system (pSerOTS-C1* (V70) 188537 ). These reagents... with the optimized phosphoserine orthogonal translation system ( 68292 ), they were able to demonstrate...central position. Introducing Hi-P for screening phosphorylation-dependent protein-protein interactions. The...

...pmiRFP703-Clathrin Clathrin-coated vesicles Clathrin miRFP703 Vladislav Verkhusha 21741 Clathrin-LCa-EYFP...Helenius 70217 pEGFP-GFP11-Clathrin light chain Clathrin-coated vesicles Clathrin light chain GFP Bo Huang...Huang 54040 mEmerald-Clathrin-15 Clathrin-coated vesicles Clathrin mEmerald* Michael Davidson 79997 pmiRFP703...TdTomato IX Membrane Palmitoylation TdTomato Connie Cepko 22479 FUmGW Membrane Palmitoylation GFP Connie Cepko...Membrane Palmitoylation sequence from GAP43 EGFP Connie Cepko 61099 Lck-GFP Membrane Palmitoylation sequence... EGFP Toren Finkel 14435 mRFP-Clc Clathrin-coated vesicles Clathrin, light polypeptide mRFP Ari Helenius...Clathrin-LCa-EYFP Clathrin-coated vesicles Clathrin, light polypeptide EYFP Xiaowei Zhuang 14437 mRFP-Rab5 Early endosomes...

...produces a colored product that accumulates over time which is relative to the amount of enzyme present...quantify relative amounts of protein. Includes: Western blot ELISA Capture - used to isolate specific ... one type of antibody. Monocultures of B cells isolated from animals immunized as described above are ... regions of the antibody to be independently manipulated. Antibodies produced this way are called recombinant... used while suspended in the original media or isolated and placed in a buffer. Antibody Structure Figure...antibody fragments (shown in Figure 2) to completely unrelated molecules. Examples include: scFv - Single-chain...instead of modified antibodies and are not closely related to antibodies. Monobodies - Monobodies are synthetic...

...target in the HR template, you usually would need to make mutations of the HR template to avoid this donor...my HR template an issue? Yes. If you have intact protospacer+PAM sequence within the HR template, it will... that if a CRISPR is used to engineer a locus, isolate single cell colonies, then genotype them, it is...Homologous Recombination (HR) FAQs Can you summarize HR template vector design guidelines? A few notes below are...you should consider using ssDNA oligo as donor template, it's usually more efficient than plasmid donor...1ul of 10uM oligo stock for each well on 24-well plate, co-deliver into the cells with the Cas9/sgRNAs ...with Cas9/sgRNAs vector, for one well in 24-well plate. A good example/reference is this Cell paper: http...

...enhanced signaling of mTOR, a key metabolic regulator, correlates with poor prognosis in many cancers....RRAGB RRAGC RRAGD Ras-related GTP binding Raptor Also known as RPTOR; Regulatory associated protein of...arget o f r apamycin (mTOR) is a key metabolic regulator controlling cell growth and proliferation...determine the nutritional state of the cell and regulates the switch from anabolic to catabolic processes...Enhanced signaling through either mTORC1 or mTORC2 correlates with poor prognosis in many cancers, including...Akt and MAPK/ERK, both of which are commonly dysregulated in cancer. Loss of the tumor suppressor p53 ... caused by inactivating mutations in negative regulators of mTORC1. The hyperactivation of mTORC1 in cancer...

...(ras related) RALBP1 RalA binding protein 1 RALGDS Ral guanine nucleotide dissociation stimulator RAPGEF...have an active (GTP) and inactive (GDP) state, regulated by the addition and removal of a phosphate group...GTPases play prominent roles in signaling pathways regulating cell migration, survival, adhesion, growth, and...differentiation and survival. Ras is essential for regulating the growth of the cell, however when mutations...for clarity and does not indicate a specific relationship. The content and map for this page were generated...EIF4EBP EIF4EBP1 EIF4EBP2 EIF4EBP3 Eukaryotic translation initiation factor 4E binding protein ERBB2 Erb-b2...

...cortical interneurons under the control of the E2 regulatory element Jordane Dimidschstein 135630-AAV9 pAAV-S5E2...cortical interneurons under the control of the E2 regulatory element Jordane Dimidschstein 135630-PHPeB pAAV-S5E2...cortical interneurons under the control of the E2 regulatory element Jordane Dimidschstein 135631-AAV1 pAAV-S5E2...cortical interneuronsunder the control of the E2 regulatory element Jordane Dimidschstein 135631-AAV9 pAAV-S5E2...cortical interneuronsunder the control of the E2 regulatory element Jordane Dimidschstein 135631-PHPeB pAAV-S5E2...cortical interneuronsunder the control of the E2 regulatory element Jordane Dimidschstein 135632-AAV1 pAAV-S5E2...cortical interneuronsunder the control of the E2 regulatory element Jordane Dimidschstein 135632-AAV9 pAAV-S5E2...

...include histone acetylation by p300, histone demethylation by LSD1, cytosine methylation by DNMT3A or MQ1...changes, researchers use ssDNA or dsDNA repair templates with homology to the DNA flanking the DSB and ...edit close to the gRNA PAM site. When a repair template is present, the cell may repair a DSB using homology-directed...Cut Base Edit Nick Prime Edit Transcriptional Regulation Activate Interfere Epigenetics RNA Targeting ...100 bp window. This technique creates diverse populations of mutants for directed evolution. Plasmid Gene...Nickase mutants can also be used with a repair template to introduce specific edits via homology-directed...primer binding site and the desired edits on an RT template. Plasmid Gene/Insert Promoter Selectable Marker...

...NTR 2.0/MTZ-based cell ablation system that allows sustained cell loss and ablation of previously...localization in high-throughput. MAZERATI platform - Leonard Zon Lab. A platform to rapidly model potential cancer...embryo and larvae, it is easy to culture large populations that are amenable to genetics, high-throughput...emerged as a model system for human disease and translational research, including the study of cancer, inflammation... enabling CRISPR/dCas9-interference (CRISPRi) modulation of enhancers and non-coding RNAs in zebrafish...organism lineage tracing by combinatorial and cumulative genome editing - Jay Shendure Lab Simultaneous...droplet reporters - Richard White Lab. An imaging platform to study lipid dynamics in vivo. Bimolecular fluorescence...