...when using them. There are suspected cases of insertional mutagenesis in humans (1) and AAVs are non-enveloped... in the tissue/what cell type? In the CNS, for instance, there are marked differences among serotypes ... second strand synthesis before gene expression begins. ScAAV avoid this rate limiting step and can immediately...complexes to study neural function. Often the reporter proteins associated with these complexes are so robustly...References 1. Russell, David W. "AAV vectors, insertional mutagenesis, and cancer." Molecular Therapy 15.10...vectors: packaging capacity and the role of rep proteins in vector purity." Human gene therapy 18.2 (2007...

...image. You could probably also do Western blotting against GFP expression, but I didn’t. 2. In the original...data not shown). Pro-Tips: 1.Designing primers against genomic DNA to amplify the gRNA targets was challenging...digestion products (that would co-migrate on a gel) instead of two different sized digestion products (which...make a figure with the data). Using acrylamide instead of agarose gives much sharper bands. I compared...you are analyzing short DNA fragments, run PAGE instead of agarose to get sharper bands. Conclusion Overall...C, Vogelstein B, Bunz F. Genetic knockouts and knockins in human somatic cells. Nature Protocols 2007;...

... selectable markers, tags, origins of replication, and fluorescent proteins that are already included ...backbone sequences, and any known sequences for the insert. The Snapgene desktop software allows us to quickly...using the Snapgene desktop software. If a plasmid contains a region with a high amount of guanine and cytosine...digest to confirm the size of the plasmid and the insert. By using the “Simulate agarose gel” option with...

... antibodies drive an effective immune response against a pathogen. The Fc domain creates a link between...expressing Fcγ receptors binds to IgG and release cytotoxins that kill the target cell. If the pathogen is...and eosinophils, where the eosinophils release cytotoxins via degranulation.  Antibody-dependent cellular... functions and all that antibodies can do! For instance, some Fc receptors, once bound to an antibody,...Inhibitory Fc receptors modulate in vivo cytotoxicity against tumor targets. Nat Med. 2000 Apr;6(4):443-6. doi...

...this post we'll focus primarily on antibiotics against Gram negative bacteria. In future posts, we'll ...bacteria, and general working concentrations. For instructions on how to prepare antibiotic stocks, see Addgene's... as a marker for successful cloning. In these instances, the cloning vector typically has two separate...that are only resistant to the antibiotic that remains intact. Tips and tricks from the bench Use fresh...Tetracycline is used. Keep in mind that some E. coli strains have natural antibiotic resistances, so make sure...

...stochastic and combinatorial expression of fluorescent proteins is only possible at low copy number ranges, resulting...2018) (Figure 1). Once introduced, fluorescent proteins were best imaged after tissue clearing with SeeDB2...expression levels. Clearing large tissues - Fluorescent proteins are very stable in SeeDB2. However, SeeDB2 is ...strategies for combinatorial expression of fluorescent proteins in the nervous system." Nature 450.7166 (2007)...labeling of neuronal circuits with fluorescent proteins and chemical tags." eLife 7 (2018): e40350. PubMed...

...-free system, capable of producing fluorescent proteins and enzymes. Image from Moore et al., 2021. ...through monitoring the expression of fluorescent proteins and oxytetracycline enzymes. Next, this system...libraries: Cas13X and Cas13Y, two compact Cas13 proteins found in the metagenome datasets from hypersaline...messenger-RBP sgRNA Library targets human mRNA binding proteins with 10 guide RNAs per gene. The lab modified ...also target core operons in other pneumococcal strains. The BARBEKO sgRNA library uses CRISPR cytosine...

...recombinant proteins at high levels. In a case where clinical applications of recombinant proteins are necessary...cells may be particularly useful due to their intrinsic properties. HEK-293 cells are tumorigenic, ...cell line’s ability to faithfully produce these proteins and its relatively low expression of native channels...Cell Line as a Platform to Produce Recombinant Proteins and Viral Vectors. Front Bioeng Biotechnol. (2021...

...Every year, the Rare Genomics Institute sponsors a global competition for researchers working on rare...He says that the award from the Rare Genomics Institute and Addgene will make a substantial difference... (top right) and his colleagues at the Wistar Institute in Philadelphia, PA will put their award to work...Joshua Wang (bottom right), also of the Wistar Institute, says that the lab got interested in XP out of...Resources Find out more about the Rare Genomics Institute and the BeHeard Award!    ...

... Biological repositories that distribute mouse strains, plasmids, cell lines, and much more have been ... Bioresources Cell Bank, part of the National Institutes of Biomedical Innovation, Health, and Nutrition...19 plasmids and resources! Case study 3: Mouse strains Reagents developed during the SARS-CoV pandemic...The Jackson Laboratory, a nonprofit research institution, has been able to rapidly propagate this strain... Aliyah Weinstein...

...based on your downstream application, time, and instrument availability. UV absorbance Using UV absorbance...acids (DNA and RNA) absorb maximally at 260 nm. Proteins on the other hand absorb best at 280 nm and organic...you can compare the fluorescence of your sample against this curve to quantify your DNA prep. Though this...this method doesn’t tell you about contaminating proteins or chaotropic salts. Capillary electrophoresis...read with a standard ELISA reader if no other instruments are available. Some last reminders about quantifying...

... when a researcher decides on a new project and begins by trying to reproduce someone else’s result. Then...time that they thought would be moving forward instead trying just to get going. It’s like being stuck... in science needs both a job and funding, so institutions and funders, to a large extent, hold the keys...is clearly room to do more. If we could really instill a culture of rewarding good scientific citizenship...UK and then a postdoc at The Scripps Research Institute in La Jolla, California. She joined Elsevier after...

...Wang, Y., Missinato, M. A., Onuoha, E., Perkins, L. A., Watkins, S. C., . . . Bruchez, M. P. (2016). A ...you can do just that.  Photosensitizing dyes and proteins have been around for awhile (check out this review...produced by photosensitizers inactivate target proteins or cells while having little effect on neighboring...suggests that it could be used with these fluorescent proteins. He et al also compared MG-2I-dL5** head-to-head...

...differentiated cells simply by expressing four proteins Oct4, Sox2, Klf4, and cMyc (the so called OSKM...Yamanaka's work has been profound, and this is a rare instance in which "profound" seems like an understatement...degeneration; Laurenz Studer discussed his work with Parkinson's Disease; Hideyuki Okano presented ground-breaking...clinical trials for ALS.   I have an uncle with Parkinson's, a half-brother who is a quadriplegic due to ... Eric J. Perkins...

...searching the interwebs for lab resources, and instead conveniently provide them on your smart phone. ...holds the Gibco Cell culture manual and basic instructional videos. The search function in the manual just...that require restriction enzymes or PCR. The app contains a double digest tool that is only helpful for ...for NEB restriction enzymes since the instructions use NEB buffers. Nonetheless, if you are setting up a...draw an amino acid or peptide for some reason. It contains all twenty amino acids plus selenocysteine and...

...in monoallelic diseases In 2013, a team led by Jinsong Li from the Chinese Academy of Sciences found that...independent team led by Hans Clevers at Hubrecht Institute in The Netherlands used the CRISPR/Cas9 genome...handle on how cleavage sites are repaired.” Those insights will be critical for scientists’ ability to influence...addition, the current work in developing anti-CRISPR proteins aid the limitation of these mutations, to control...s Disease.” Journal of Alzheimer's disease & Parkinsonism. https://doi.org/10.4172/2161-0460.1000439 6...

...second GFP antibody?  Similarities:  Generated against full length GFP and converted to recombinant form...Isotype IgG2a Differences:  Distinct variable domains that recognize different epitopes on GFP Monoclonal...light over the last decade, but the fluorescent proteins (FPs) necessary for many of these applications...consider hfYFP and LSSmGFP.     Fig. 4: hfYFP retains folding and fluorescence in high molarity GdnHCl...Campbell, B.C., et al. Chemically stable fluorescent proteins for advanced microscopy. Nat Methods 19, 1612–...

...sections, while still creating scarless plasmid inserts.  Want to Learn More about DNA Assembly? Check...polymerase used in the amplification. This tool allows instant updates as fragments are added, deleted, or reordered...(sgRNA). Using only one single-stranded oligo, instead of the more typical two oligos, is more cost effective...linearized plasmid with a synthetic oligo that contains a region of homology and any desired edits to ...amplified with overlapping regions of homology and inserted into a linearized cassette, ready for transformation...

...certified health insurance executive (CHIE). He is a member of both the Internal Institute of Auditors and... Directors brings complementary experience and insight to the now 10 member board at Addgene. Let’s meet...at both MetLife & Ernst & Young, LLP, auditing insurance, finance and high-tech industries. He has earned...Addgene blog Learn more about about Addgene in our "Inside Addgene" blog series Subscribe to the Addgene blog...

...AAV? A: One potential strategy is to use split-inteins to mediate trans-protein splicing. Note, however...possible to make an efficient AAV prep for a 4.2 kB insert with the CMV promoter using the AAV-BR1 capsid?...bringing total genome length to ~4.7kB.  If your insert alone is 4.2 kB and CMV promoter+enhancer is 0.5...experiments using PHP.eB in the brain give some insight into codelivery efficiency at different doses. ...tropism to other serotypes, but reproducible success remains elusive.  There has also been a lot of effort in...