...Epub 2016 May 2. PubMed PMID: 27136078. Find plasmids from this publication at Addgene. 2. Swarts, Daan... be stable in the cell. gDNA guides will also need to be transfected into the cells of interest. In contrast...unable to show TtAgo-mediated cleavage in mammalian cells, likely due to high temperature requirements. To...plasmid and genomic DNA in a variety of mammalian cell lines, with an efficiency similar to that of Cas9...

...starting day 2 after transfection.     Fig: 2: Expressing mARGAna in MDA-MB-231 cells. (a) Plasmid...expression. When engineering a cell line, cotransfect the mARG cassettes 2:1 (A:N-V) with PiggyBac transposase...producing cells. After FACS, return the cells to doxycycline-free media and expand the cells. Use a subset...white inclusions in cells with GVs via phase contrast microscopy, GVs visible in cells via TEM, and purified... heterogeneity required single cell screening to isolate a clonal cell line capable of producing ultrasound...expression homogeneity, as the cells could be batch-sorted to isolate a polyclonal cell line with strong gas vesicle...transient transfection in rtTA+ cell lines (such as HEK293-TETOn cells), or they can also be integrated...

... Plant Methods. BioMed Central, 2(1), p. 19. doi: 10.1186/1746-4811-2-19. PubMed PMID: 17087829. PubMed...PMID: 24032037. PubMed Central PMCID: PMC3759027. 2 Harrison, S. J. et al. (2006) ‘A rapid and robust ...incubated at stationary phase for too long, the cells in your culture will no longer be viable. The time...graduate student at Stanford University. She studies cell fate maintenance in the stomatal lineage and has...

...daughter cell, instead of partitioning each type of plasmid into a separate daughter cells (Figure 2) (Ebersbach...to plasmid loss (Diaz et al., 2015).  Figure 2: The partitioning problem. For low copy plasmids...Plasmids. Microbiology and Molecular Biology Reviews 62(2): 434–464. Pubmed PMID: 9618448 Diaz R, Rech J, Bouet...copy number in the cell is high, but allows replication when copy number in the cell falls too low (Novcik...system to partition themselves to each daughter cell during cell division. They don’t simply rely on chance...eukaryotic cells). The partition NTPase is recruited and “walks” each plasmid to a separate daughter cell. When...from the cell with the same probability, or asymmetrical, where one plasmid lost from the cell at a higher...

...vector:insert ratio is used in the DpnI digestion and 2 µL of digest product are used in the transformation...Biology 38 (2016): 145-154. PubMed PMID: 27391134. 2. Li, Chaokun, et al. "FastCloning: a highly simplified...properly. Directly transform chemically competent cells with the digested mixture. There is no need for ...purification before transformation. After transforming cells, you should perform colony PCR with vector specific...interested in protein science.     References: 1. Celie, Patrick HN, Annabel HA Parret, and Anastassis Perrakis...

... than control, 3; same as control (Cetn1/sgRNA1), 2; darker than control, 1; very dark). Brighter fluorescence...you can hold off on genotyping until the pups are 2-4 weeks old and use the ear tag method (creating different...PMID: 24253446. PubMed Central PMCID: PMC3875854. 2. Mashiko, Daisuke, et al. "Generation of mutant mice...expanded since its adaptation for use in mammalian cells in 2012-2013. Researchers are now using this system...plasmids containing sgRNA sequences into HEK293T cells. The fluorescence intensity was classified into ...is then a simple matter of transfecting HEK293T cells with your modified pCAG-EGXXFP plasmid along with...incubation, you assess the level of fluorescence in your cells under a fluorescence microscope. The higher the ...

...presented in a series of quick (10 minute) or very quick (2 minute) talks so we got to hear from everyone. Addgene...evolutionary tree at least 6-7 times and on at least 2 separate continents. Mimulus (Andrea Sweigart) Monkeyflowers...Organisms, Part 1 Five Popular Model Organisms, Part 2 ...Xenopus frog cell lines to develop robust methods for frog gene editing. They edit in a cell line, then ...is the smallest photosynthetic cell in existence, but most abundant cell on earth (twice the human biomass...only 1,700 genes total). Hydra (Celina Juliano) This is a known stem cell and regeneration model. Little...Mormyridae (Jason Gallant) Weakly electric fish are excellent systems to study for convergent evolution because...

...into the several steps outlined below and in Figure 2. For a deeper dive into the steps and techniques, ...mounting sample on slides, and confocal   Figure 2: High-level overview of an indirect immunofluorescence...recognize the species of your primary antibody and 2) is conjugated to a fluorophore that suits your experiment...interest live within a cell,” “Does this disease change the architecture of my cells,” or “How does this ...IF), is an immunoassay that brings to light the cellular world. The technique allows you to ask questions...this mutation impact the types of cells found in my tissue.” It is based on the same principles as other ... visualize a target of interest within an intact cell, tissue, or, in some cases, a whole organism. But...

...Endonuclease of a Class 2 CRISPR-Cas System. Cell 163:759–771 . https://doi.org/10.1016/j.cell.2015.09.038 Find...is unique compared to the rest of the genome and (2) the sequence is located just upstream of a Protospacer...pyogenes Cas9 variants with varying PAM sequences, 2) the use of Cas9 homologs derived from species other...are capable of cleaving genomic DNA in mammalian cells and zebrafish embryos, and they can be used to modify... adapted for targeted RNA cleavage in mammalian cells. The REPAIR (RNA Editing for Programmable A to I...reveals specificities of Cpf1 endonucleases in human cells. Nat Biotechnol 34:863–868 . https://doi.org/10.1038...specificities of CRISPR-Cas Cpf1 nucleases in human cells. Nat Biotechnol 34:869–874 . https://doi.org/10.1038...

...native enzyme, which are summarized in Table 2 below.  Table 2: Properties of native luciferases Organism...luciferase assays require cell lysis as the most efficient means to disrupt the cell membrane and deliver ...miRNAs for effectiveness. Analyze Cell Signaling Pathways Known cellular signaling pathways can be investigated...luciferase as a genetic reporter in plant and mammalian cells. Luciferase assays have since become a gold standard... serves as an indicator of transcription within cells, where detection of the reporter protein or its .../Phototoxicity Not susceptible Susceptible Subcellular Imaging Improving Well-established High-throughput... into the desired mammalian, plant or bacterial cells. Expression of luciferase as the reporter gene is...

...the reporter mRNA is labeled by PCP-mCherry (Figure 2). The 3’ UTR also contains a CAAX sequence to bind...PMC2746483 Find ribosome profiling plasmids at Addgene. 2. Halstead, James M., et al. “Translation. An RNA biosensor... of all of the mRNA bound-ribosomes in a cell. When cellular mRNAs are digested with ribonucleases, ribosomes...Regulating translation is key to cellular function, especially during development or stress. With ribosome...explore the complexity of translational control in cellular physiology. Ribosome profiling Ribosome profiling...the effects of 5’ UTR variants in translation of cell cycle protein Emi1, showing that ~80% of transcripts...powerful tools for studying translation in various cell types and physiological states. References 1. Ingolia...

...Mammalian genomic DNA is not just a 2-D linear molecule snaking through our cells’ nuclei--our chromatin is comprised...target sites for editing. PAM sequences can be from 2-6 base pairs in length and follow the DNA sequence...plant protein TIR1 in non-plant cells, which interacts with the cells’ endogenous ubiquitin ligase, the...translate AID to yeast, as well as mammalian and human cell lines (Nishimura et al., 2009, Natsume et al., 2016...based method to tag endogenous proteins in HCT116 cells with mAID. Using this method, the lab was able to... protein of interest to create conditional human cell lines. Lastly, the Kanemaki lab demonstrated that...the target sites adjacent to an NG PAM in human cells. The increased versatility of the engineered SpCas9...

...improve solubility and stability (Figure 2).      Figure 2: Crystal structures of CRBNmidi alone...fluorescent tags in Caenorhabditis elegans. Genetics, 228(2), iyae126. https://doi.org/10.1093/genetics/iyae126...useful addition to the toolkit for visualizing cellular processes in C. elegans: mScarlet3 for applications...biologically relevant forces at focal adhesions in living cells in several different contexts.   Figure ...substrate furimazine. Right: Demonstration of PILATeS. Cell adhesion to a PILATeS-functionalized coverslip creates...

... qPCR is that the results can vary by a factor of 2. This means that if you set up two identical assays...amplitude measured for each droplet in each well (Figure 2). A clean ddPCR should have a clear separation between...as low as 1C/s. Increasing the elongation time to 2 minutes and the denaturation time to 1 minute has ...used in the detection of rare sequences and single cell analysis. It is also being used in the detection...

...represents the time and intensity of the cell’s illumination (see Figure 2). H describes the maximum signal strength...most people use that setting. Figure 2: As the cell passes through the laser beam and causes it...proportion (while H and W do not; see Figure 2), single cell events will show up in a straight line one ...Many cell types can be found in the tumor microenvironment (tumor cells, immune cells, blood cells and ...about 53% of my cells are dendritic cells and ~13% are CD8+ T cells. Around 34% of the cells are neither ...neither of the two cell types (likely B cells and others). Almost no cells stain positively for both markers...measure your cells of interest. In many experiments you’ll be working with a heterogeneous cell population...

...host range plasmids. FEMS Microbiology Letters, 348(2), 87–96. https://doi.org/10.1111/1574-6968.12241  ...mobilization of broad host-range IncQ plasmids. Plasmid, 62(2), 57–70. https://doi.org/10.1016/j.plasmid.2009.05.001...negative hosts. Biotechnology and Bioengineering, 106(2), 326–332. https://doi.org/10.1002/bit.22695  Schmidhauser...ability to establish and maintain itself within host cells.  The plasmid has multiple ori that are functionally...eutrophus, Bartonella bacilliformis, Bordetella spp., Brucella spp., Caulobacter crescentus, Escherichia coli...

...panning and scFv characterization.    Figure 2: Phage display starts with a pooled library of scFvs...the short epitope GCN4, also called a scaffold; and 2) a GCN4 binding scFv that’s fused to GFP. When the...Fv (Variable Domain) (scFv) Fragments. Antibodies 2:193–208 . https://doi.org/10.3390/antib2020193  Khantasup...scFvs and T cell receptors and are expressed on T cells to make CAR T cells. The extracellular scFv portion...Bispecific T-cell engagers (BiTE®s), bind CD3 on T cells and a tumor-specific antigen, it brings T cells to a ...recognizes an antigen of interest while the intracellular T cell receptor portion of the CAR facilitates ...scFv part of the receptor can activate T cells to kill cancer cells. scFv’s small size also allows for their...

... Figure 2: Ironing boards. Good as a WFH desk, and as a video control center.   Cycle 2: Zooming ...ironing board, giving it the perfect height (Fig. 2). We recorded video using a simple, external video...Zooming Forward Design: We produced a video celebrating Massachusetts Nonprofit Day, which was the perfect opportunity...  Quintin Marcelino...

...immunosurveillance." Cancer cell 30.1 (2016): 147-160. PubMed PMID: 27411589. 2. Todoric, Jelena, Laura Antonucci...represent a favorable state for tumor progression (Ref 2). Future treatments may need to target this immunosuppressive...cancers. It was inspiring to participate in the 2017 Cell Symposium on Cancer, Inflammation and Immunity and...recent findings in basic immunology, cancer-immune cell interactions, cancer immunotherapy, as well as new...immunosurveillance. The presented study analysed a model of hepatocellular carcinoma, a cancer which can develop from ...example the upregulation of the immunosuppressive cell surface receptor PD-1. This immunosuppressive environment...follow the interactions between tumor and immune cells in real time. Addgene depositor Professor Max Krummel...

... variation (CNV) analysis of the U2OS cells.   Figure 2: CRISPR-Sirius tricolor labeling. (A) ...that the signal to noise ratio can be increased from 2-fold (by CRISPRainbow) to 3-fold with CRISPR-Sirius... to date for visualizing genomic loci in living cells. Previous iterations of genomic loci visualization...labeling system to visualize chromosomal loci in human cells (Ma et al., 2015). To do so, they fused a different...visualization of sites on different chromosomes in living cells, the system requires high-copy chromosome-specific... aptamer system to visualize the gRNA in living cells. Broccoli aptamers are nucleic acid sequences that...production needed to deliver CRISPR-Sirius into cells. They named this guide sgRNA-Sirius-8XMS2 which ...