...Cas9 (dCas9) or a Cas9 nickase (nCas9) to avoid introducing double-strand breaks. The only things base editors..., the team noticed that this editor sometimes introduced C → G edits at certain positions within the editing...during cytosine-to-uracil editing, and instead introduced its opposite, a uracil DNA N-glycosylase (UNG...end joining (NHEJ). To mitigate this, the team introduced the enzyme HMCES, either as a fusion construct...ever. References and Resources Glossary We introduced a lot of terminology in this post! Here’s a quick...

... promoters are upstream or 5’ of the gene they control. Promoters can be organism or tissue type-specific...different levels of expression. For example, CAG is a strong promoter that drives very high, ubiquitous expression...recombinase, are used to spatially and temporally control transgene expression. Lox sites are directional...availability of Cre, lox sites lets researchers control the spatial and temporal activation of a transgene...

...increased yields in adenoviral (Coroadinha, 2006) and retroviral production systems (Shen, 2011). To our knowledge...also improved yields of the AAV2 derivative, rAAV2-retro, but had no effect on AAV5 yields. We further show...preparation. Through sugar detection assays, in vitro transduction, and silver stain approaches we show...Effect of osmotic pressure on the production of retroviral vectors: Enhancement in vector stability." Biotechnology...

...group optimized their initial system by using a stronger promoter and different biochemical secondary energy...cell free system allows for a relatively simple, controlled “one-pot” reaction capable of reproducing useful...codons, splice acceptor and donor sites, and introducing premature stop codons.    New from the viral... and 5HT sensors New AAV tools to selectively control parvalbumin interneurons from the Dimidschstein...

...gene-editing protein - a DNA-cleaving Argonaute from Natronobacterium gregoryi, or NgAgo for short. Addgene has ..., showing that this protein could cleave DNA in vitro, directed by phosphorylated DNA guides. Unfortunately...RNA guides must be expressed from a plasmid or in vitro transcribed, and changes in gRNA secondary structure... Han. “DNA-guided genome editing using the Natronobacterium gregoryi Argonaute.” Nat Biotechnol. Epub ...

...PubMed PMID:29109157. Nanobody toolkit to monitor retrograde transport at the cell surface Article contributed...developed a set of functionalized nanobodies to study retrograde transport from the cell surface. Their toolkit...treated with biotin–phenol and H2O2, APEX2 labels electron-rich amino acid side chains (e.g. Tyrosine) with...biotin-labeled proteins can them be analyzed by mass spectrometry allowing a researcher to specifically identify...

...translated into proteins, or, in the case of retroviruses, reverse transcribe their gRNA into DNA, which...is. Thankfully, through the use of engineering controls — changes made to the virus that eliminate or ...viral vectors to reproduce only in experimentally controlled conditions, making them far safer to work with...they’re derived from: AAVs, lentivirus, gamma retrovirus, and adenovirus. And now that you know what viral...

...epitope tags? Epitope tags are short peptides introduced at the N or C terminus of a protein that are ...hundred amino acids, depending on the tag, and are introduced via knock-in at a genomic locus or more commonly...should be used with caution or not at all. The introduction of a tag at the N or C terminus of a protein...  Susanna Stroik...

...chemically or electro-competent E. coli - excess salt causes arcing during electroporation which would ...sequenced for potential positive clones. Once you introduce your 3’-end “A” overhang insert, the magic of ...would cause the electroporation to fail). When incubating at room temperature, it is not recommended that...

... 10. Plasmids 101: Control Plasmids Need help thinking about the types of controls you’ll need for your...provides researchers with many different ways to control gene expression. Get the full scoop from Addgenie...Addgenie Chari Cortez guides us through some common controls and why you might need them. Happy Reading!   ...

...pairs from intergenic regions, intronic regions, and a pericentromeric region. All pairs were easily visualized...CRISPRainbow. Both tools were targeted to the FBN3 intronic repeat which contains 22 copies of the gRNA target...locations of the subtelomeric region of the pericentromeric region (PR1), the p-arm (T1), and the q-arm...

...later, it was shown that putting the operon under a strong promoter results in high R-body expression  (Schrallhammer...bodies could also perform as actuators in microelectromechanical systems (MEMS) devices. They could be functionalized...of armor to protect their host from predation (Petroni et al 2000). With a better understanding of how... Plasmids from This Publication at Addgene 4. Petroni, G., Spring, S., Schleifer, K.-H., Verni, F. & ...

...Xin hypothesized that she could make enzymes controllable by tethering them to Dronpa, a fluorescent protein...their ability to be precisely spatio-temporally controlled as a key advantage over other inducible enzyme... sugars (including lactose), and a lac operon controlled GFP. When the enzyme breaks down the HMO of interest...Photosactivatable Fluorescent Proteins Use Light to Control CRISPR Genome Editing Choose the Brightest Fluorescent...

...abstracts comparing the mortality rates between two controversial health issues. One group read the text from ...design or by their preconceived ideas about the controversial medical issues. Participants gave significantly...methodologically flawed, and biased. As a result, the control abstract comparing two fictional medical issues...and education have more polarized beliefs on controversial science topics. Proceedings of the National ...

...The gRNAs targeting EGFP can be used as positive controls for gene editing or gene activation, while the...What you don't need to do is spend your time troubleshooting reagent production, because that's a distraction...from your primary goal. Getting these quality-controlled reagents from Addgene can greatly expedite a ...Research: A Primer for Beginners 5 Tips for Troubleshooting Viral Transductions Additional Resources on...

...digest as depicted in Lane 2. Controlling methylation Finally, you can control methylation by altering your...only the non-mutated and methylated template is destroyed leaving behind a pool of mutated plasmids which... with the Latest Cloning Techniques Learn to Troubleshoot Your Sequencing Results Use REPLACR Mutagenesis...

...unique editing tasks, classic Cas9 nuclease, which introduces double stranded DNA breaks, can be expressed ...Gαi-coupled) DREADD fused to mCherry, all under the control of the human synapsin promoter. Purpose of this...mCherry can be used to induce neuronal silencing in vitro and in vivo (Krashes et al., 2011). The various ...Rumpel S. Analysis of transduction efficiency, tropism and axonal transport of AAV serotypes 1, 2, 5, ...

...antibodies. The introduction of antibody fragments, such as nanobodies, has introduced new options for...Antibodies 101: Introduction to Immunofluorescence Antibodies 101: ELISAs Antibodies 101: Introduction to Antibodies...

...antibody, you’ll want to use the same positive control across experiments to be aware of signal strength...you’ll want to be sure to choose the appropriate controls to be sure the staining you are seeing is real...real. It’s important to include a negative control using serum from the same species your polyclonal antibody...antibody was generated in or a polyclonal isotype control, to account for nonspecific binding. Now that you...

...localization. Surprisingly our observations at the electron microscope showed us that, in monkey neurons, ...of the haemagglutinin of the influenza virus, introduced to the field of fusion proteins more than thirty... of interest the membrane.   Figure 1: Electron microscope images showing ultrastructural localization...protein of interest, an alternative could be bicistronic expression of the fluorescent protein with the...