We narrowed to 16 results for: URE

...addition of a structured motif to enhance pegRNA stability PEmax — optimized PE architecture; increased ...synthesized (Figure 8A), then cloned in a pooled format into lentiviral transfer vectors (Figure 8B). Libraries...highest levels of single-gene activation (Figure 9D) Figure 9: Overview of CRISPRi/a. In bacteria, activating...Berkeley. Plan Your CRISPR Experiment Resources Figures All figures were created using BioRender.com (Link opens..., N., Ishitani, R., Zhang, F., & Nureki, O. (2014). Crystal Structure of Cas9 in Complex with Guide RNA...vivo genome editing using Staphylococcus aureus Cas9. Nature , 520 (7546), 186–191. PMID: 25830891 Rauch...changing the target sequence present in the gRNA. Figure 1: Overview of the basic CRISPR mechanism. CRISPR...

...isolated and placed in a buffer. Antibody Structure Figure 1: Structure of an antibody A standard antibody is...Antibodies can also be produced in cell cultures using hybridomas, cell cultures created by fusing B cells with...form a “Y” shape, as shown in Figure 1. The two arms of the Y structure are responsible for binding the...Monoclonal antibodies are generated from cultured B cells, where each culture should produce the same antibody...Diabodies can be used for assembling protein nanostructures. Figure 2: An antibody (IgG, far left) compared... a signaling molecule. Figure 4: A direct sandwich ELISA, which uses a capture antibody and a primary ...In a sandwich ELISA, a capture antibody, bound to a surface, is used to capture the protein of interest...

...Table 1. Figure 4: Third-generation lentiviral plasmids. Created with BioRender.com. Feature Second-generation... chromatin architecture through large-scale mapping of lentiviral integration sites. Nature Protocols ...to continue their lifecycle and replicate. This feature can be harnessed for stable delivery of various... typically ranges from 8–10 kb, encoded on RNA (Figure 1). This RNA is reverse transcribed into the provirus...have been removed or mutated for increased safety. Figure 1: Wild-type lentivirus genome. Created with BioRender.com...largely intact. First-generation plasmids include (Figure 2): Transfer plasmid — contains transgene and wild-type...envelope removed Envelope plasmid — contains env Figure 2: First-generation lentiviral plasmids. Created...

...replication and also act as signals for packaging. Figure 1: Wild-type AAV genome. Created with BioRender.com...do not trigger a strong immune response. These features have made AAVs the preferred choice to be used...components, see the AAV Plasmid Elements table . Figure 2: Plasmids required for rAAV vector production...methods (such as a Gateway destination vector), so be sure to confirm your chosen plasmid is compatible with...available in your lab. When cloning your plasmids, be sure to use recombination deficient bacterial strains... the ITRs found in transfer plasmids. This will ensure that the repeats will be maintained and often results...and instead provided by the chosen cell line to ensure safety and prevent the potential formation of replication-competent...

...elements are summarized in the table below. Figure 2: General structure of a plasmid. Plasmid Element Description...bacterial cultures with antibiotics, see Addgene’s protocol on inoculating bacterial cultures . Antibiotic...for proteins are transcribed and processed into mature messenger RNA (mRNA), which is then translated ...study DNA fragments of interest, such as genes. Figure 1: Creation of recombinant DNA. Working with Plasmids... organisms, including plants, worms, mice, and cultured human cells. Have diverse applications — Although...one unique restriction enzyme recognition site (Figure 2). These elements allow for the propagation of...propagation of these plasmids through cell division. To ensure the retention of plasmid DNA in bacterial populations...

...different optimal editing windows. Be sure to refer to the literature associated with your chosen editor....activity. Be sure to sequence the genomic region you intend to target in your samples to ensure there are...components (Figure 2), and the optimal system will likely depend on your cell type. Figure 2: Different...(Cas enzyme and guide RNA) for your experiment (Figure 1). You will decide how to express Cas9, the delivery... and finally how to validate your genetic edit. Figure 1: Flow chart describing the general framework ...and essentially function as a “knockout,” but be sure to consider potential consequences of having a non-functional...only compatible with smaller Cas enzymes, like S. aureus Cas9. For more information, see our blog post on...

...Collection page . Figure 5: Overview of AdEasy™ adenoviral vector production system. Figure is based on the...assemble icosahedral capsids and build new virions. Figure 1: Wild-type adenovirus and its genome. Genes over...which incorporate additional genomic deletions. Figure 2: First-generation rAdV vectors. Created with ...using first- and third-generation rAdV vectors. Figure 3: Second-generation rAdV vectors. Created with... challenge of the HCAdV vector production is to ensure no carryover from the HV in final viral preparations... genome or using other plasmids instead of HVs. Figure 4: Third-generation rAdV vectors and helper virus...methods (such as a Gateway destination vector), so be sure to confirm your chosen plasmid is compatible with...

...to continue their lifecycle and replicate. This feature can be harnessed for stable delivery of various...gamma-retroviruses ranges from 9–11 kb, encoded on RNA (Figure 1). This RNA is reverse transcribed into the provirus...producing viral vectors in a laboratory setting. Figure 1: Wild-type gamma-retrovirus genome. Created with...gamma-retroviral vectors, you need three plasmids (Figure 2): Transfer plasmid — contains transgene, sgRNA...infectivity for different cell types (known as tropism). Figure 2: Gamma-retroviral plasmids. Created with BioRender.com...methods (such as a Gateway destination vector), so be sure to confirm your chosen plasmid is compatible with...available in your lab. When cloning your plasmids, be sure to use recombination-deficient bacteria strains,...

...and the distal promoter (Figure 4). Figure 4: Basic eukaryotic promoter structure. TF = transcription factor...recognizes different core promoter sequences. Figure 2. Basic structure of a bacterial promoter. TSS = transcription...sense or coding strand of the transcribed gene (Figure 1). The coding strand is the DNA strand that encodes...strand that is transcribed by the RNA polymerase. Figure 1: Simplified promoter region during transcription...positions from the transcription start site (TSS) (Figure 2). The Pribnow box (TATAAT) is located at the ...is an example of a negatively-controlled operon (Figure 3). A positive-repressible operon works in the ...is an example of a positively-controlled operon. Figure 3: Lac operon regulation. Created with BioRender.com...

...guide to channelrhodopsin variants: features, limitations and future developments. Exp Physiol, 96 (1),...social dysfunction. Nature, 477 (7363), 171–178. https://doi.org/10.1038/nature10360 PMID: 21796121 Yizhar...Optogenetics Plasmid Collection . An optogenetics procedure. A channelrhodopsin, fused to mCherry, is expressed... allow the fast depolarization of neurons upon exposure to light through direct stimulation of ion channels... in excitation (depolarization) of the neuron. Feature enhancements include: Increased photocurrent amplitude...from the cryptophyte Guillardia theta ). Other feature enhancements include: Increased photocurrent amplitude...outside of the brain, rendering the experimental procedure less invasive. Different activation wavelengths...

...that form complex secondary structures. Read more in our SLIC blog post . Figure 6: Summary of LIC. Created...Read more in our Restriction Cloning blog post . Figure 1: Restriction enzyme cloning of your gene of interest...conditions. Read more in our Gateway Cloning blog post . Figure 2: Summary of Gateway cloning. Created with BioRender.com...cloning. Read more in our TOPO Cloning blog post . Figure 3: Summary of TOPO cloning. TOPO = topoisomerase...exonuclease. Read more in our Gibson Assembly blog post . Figure 4: Summary of Gibson assembly. Created with BioRender.com...Read more in our Golden Gate Cloning blog post . Figure 5: Summary of Golden Gate cloning. Created with...time- and cost-saver for labs working with yeast. Figure 7: Summary of yeast-mediated plasmid cloning and...

...explore Addgene's Chemogenetics Plasmid Collection . Figure 1: General overview of a chemogenetics experiment...muscarinic receptor and has not been used in vivo. Figure 2: DREADDs, their ligands, and signaling properties...and/or K + ions, and activates neuronal activity. Figure 3: PSAM4-based ligand-gated ion channels, their...optogenetic systems in our Optogenetics Guide . Figure 4: Luminopsins can be activated through chemogenetic... control expression of a chemogenetic reporter ensures that the reporter remains silent until a cell or...Neurosci, 37 , 387–407. https://doi.org/10.1146/annurev-neuro-071013-014048 PMID: 25002280 Strader, C. ...

...can also negatively affect data reproducibility. Figure 1: Simplified flow chart for amplifying and using...infected cell receives only one plasmid. To make sure that every plasmid is adequately represented in ...two types: positive screens and negative screens (Figure 2). Both types of screen employ a selection method...re planning to conduct a pooled library screen. Figure 2: A simplified outline for using lentiviral gRNA...cells that survive post-selection. The selective pressure must be strong enough that most of the cells die...

...conjugated to a signaling molecule, can be used to capture, quantify, and identify proteins of interest. Read...Optogenetics integrates optics and genetic engineering to measure and manipulate cells (frequently neurons) and their...optogenetics research utilize light to detect, measure, and control molecular signals and cells in order...

...Not sure where to start? Need a quick reference? Addgene's eBooks are here to help!... Collection Not sure where to start? Need a quick reference? Addgene's eBooks are here to help! 0 of 0...you need to know about plasmids, from parts, nomenclature, and uses to common promoters and cloning techniques...

... a protein-coding gene, and a terminator, as in Figure 1). Next, up to six Level 1 modules can be assembled...maintain the final plasmid after transformation. Figure 1: Overview of the MoClo Toolkit components and...synthetic biology. MoClo Plant Parts II and Infrastructure Kit Plant Expression Johannes Stuttmann 95 ...