...encapsulating the genome is majorly composed of three proteins that can be easily modified in order to facilitate...provide a large transgene packaging capacity. For instance, with a packaging capacity of about 6 kb, the ...mechanism to knock out genes as a result of an insertion, a deletion or a frameshift (Ding et al., 2014...attribute this phenomenon to the presence of terminal proteins capping the ends of linear double-stranded AdV...several biological systems. It has been used for instance in inherited disorders such as B-thalassemia, ...steatohepatitis cancer. In deficiencies of plasma proteins where Adv vector delivery is often used, the CRISPR...

...Wyss Technology Development Fellow at the Wyss Institute and Harvard Medical School. Scientists making ...drive? The simplest way to make a gene drive is to insert an endonuclease gene within its own cut site1. ...spreads through the wild population and immunizes it against the first drive. Laboratories interested in building...question), why not use them whenever applicable?Against this, consider the cost of an accidental release...accidental release could delay real-world applications against scourges such as malaria and dengue for many years... Holm, D. G. & Suzuki, D. T. Genetic Control of Insect Populations: I. Cage Studies of Chromosome Replacement...

...such as PX459, which encodes Cas9 and contains a site for inserting a custom gRNA.  While this methodology...translation delays editing What are Cas9-gRNA ribonucleoproteins? One alternative approach, which avoids many...Schier laboratories. Delivering Cas9-gRNA ribonucleoproteins  An advantage of performing CRISPR experiments...vivo applications. This technique utilizes Cas9 proteins harboring receptor ligands (C in the figure above...., et al. "Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing...

...causes the proteins and genomic DNA to precipitate. Plasmid DNA is free in solution. Proteins and genomic...the same principles: cells are lysed, RNAs and proteins are removed to leave you with DNA. This DNA is...you lyse the bacteria and denature the DNA and proteins in solution. The pH is then lowered using a renaturing...phenol chloroform extraction to remove remaining proteins and RNase. Finally, DNA is precipitated with alcohol...identical to that of commercially available kits but instead of binding DNA to silica columns, it’s bound to...

...expression while proteins needed for later experiments (cellular tracking for instance) can be controlled...Tamoxifen is added. Tamoxifen then binds to the ER-LDB domains of the fusions allowing Cre/FLP translocate to ...fluorescent cytoplasmic markers (e.g. eYFP) and synaptic proteins (e.g. postsynaptic PSD95-mcherry) are used in ...In order to analyze the function of individual proteins in neuronal circuits or synaptic remodeling, researchers...is fascinated by the question of how genes and proteins coordinate billions of neurons to form the functional...

...these experiments can be done in days to weeks instead of months to years. In the plant sciences “transient...evaluate its function. In nature Agrobacterium contains a plasmid with a T-DNA (transfer-DNA) region. ...pSB166 - pL2_pSB90_tMAS::rLUC-I::pMAS. Fluorescent proteins (FP, e.g. GFP) can be used in many ways. Often...from Agrobacterium. Test different Agrobacterium strains We used the disarmed Agrobacterium tumefaciens ...plants using syringe agroinfiltration." Recombinant proteins from plants. Humana Press, 2009. 41-50. PubMed... 5.02 (2003): 103-115. Hu, Ching-yeh, et al. "Intrinsic GUS-like activities in seed plants." Plant cell...

...yourself needing to brainstorm with a team. One major challenge with these brainstorming sessions is avoiding...with unlimited time? Another strategy for your brainstorm session is to start with something completely... students during an interview. When they were instructed to use the three-ring binder as a starting point...people to agree with the most prominent idea, and instead building an environment where everyone has their... ideas that I have found helpful in my team brainstorming sessions.  Allow the group time to think about...

...origin of replication (ori), where DNA replication begins, so a bacterial plasmid needs a compatible ori ... divide. In transient transfection, the vector remains episomal, meaning it stays separate from the genome...synthesis, and the neomycin resistance gene protects against any of them. But different aminoglycosides work...options doesn’t end there. Make sure you read the instructions for whatever antibiotic you choose to ensure...to spontaneously develop resistance to selection toxins. Auxotrophic selection is useful for producing ...researcher may choose not impose selection on cells and instead to use a vector with a reporter, such as a fluorescent...

...with higher specificity (eSpCas9 and HypaCas9) instead.  Choosing a Cas9 for site-specific modifications... effects. The SpG variant, discovered by the Kleinstiver group has a PAM of NGN (Walton et al., 2020)....system can be used with a variety of impaired Cas9 proteins (such as SpRY) with altered PAM requirements, ...facilitates recruitment of multiple activation domains to the same genetic locus via co-expression of ...epitope tagged dCas9 and antibody-activator fusion proteins. The Synergistic Activation Mediator complex, ..., R. T., Christie, K. A., Whittaker, M. N. & Kleinstiver, B. P. Unconstrained genome targeting with near-PAMless...

...pharmaceutical companies, biotech start-ups, health insurance companies, and governments on the most effective...physicians and patients, shaping discussions with insurance companies about the pricing and coverage of drugs...situations might be especially challenging. For instance, the client might need to get a large amount of...occasions, clients can be excessively demanding. For instance, a client may offer a lot of pointed feedback ...understanding of the molecular behaviors of chemicals and proteins to learn how a particular drug or medical device...therapeutic approach brings to patients, doctors, and insurance providers, and is crucial to how our clients’ ...

..., R., Weinstock, G., Metzker, M. et al. Genome sequence of the Brown Norway rat yields insights into mammalian...organism in microbiology and biochemistry. Certain strains have been specifically developed to thrive in laboratory...extensive research history and characterization. Many proteins and processes naturally found in E. coli have ...amphibians in science with early research providing insight into some of the most significant and fundamental...), 565–571. https://doi.org/10.1159/000323761 Modlinska, K., & Pisula, W. (2020). The Norway rat, from...

...unstable in vivo due to poor folding and thermal instability at 37°C (Ouellet et al., 2016) and requires a...riboswitch, metabolite binding to the riboswitch instead activates Spinach fluorescence. In the search for...identified the nucleotides that contribute to the instability of Spinach, and were able to develop an aptamer...Spinach, a miniaturized version of Spinach that retains similar levels of fluorescence when bound to DFHBI... cell imaging. Bunch of baby spinach, or BoBS, contains tandem repeats of Baby Spinach. It was developed... Aliyah Weinstein...

...to reveal even more insights. Super-duper lipid editor And if recruiting proteins to the cell membrane...photoswitchable elements into many other types of proteins. Here, we’ll highlight a few recently-deposited...et al., 2022). Stopping traffic with CIB1-Rabs Instead of using an optogenetic module to activate or recruit...such a strategy to dissect the roles of three Rab proteins during a key stage of zebrafish development (Aljiboury...(Aljiboury et al., 2023). Rab family proteins support vesicle trafficking and organelle dynamics, but ...

...reporter constructs), in functional analysis of proteins (to perform alanine scanning mutagenesis or targeted...should be resistant to Cas9 cleavage. If a plasmid contains the template, site-directed mutagenesis can be...which is dam+). Methylation deficient bacterial strains can be identified by the dam13(-) mutation – you...you’ll want to steer clear of these strains when preparing the plasmid template for site directed mutagenesis...functional regions of the plasmid including the insert. This is essential since mutations are potentially...PubMed PMID: 2448875. 4. Xia, Yongzhen, et al. "New insights into the QuikChangeTM process guide the use of...

...whether you are perusing classic papers or are brainstorming new technological innovations. Let’s take a ...blotting to screen founder transgenic animals for the inserted transgene because Southern blots have a lower ...digests. A researcher can test the orientation of an insert in only a few hours at low cost with restriction...do not typically rely on Sanger sequencing but instead use methods like pyrosequencing that are parallelized...techniques we use now may soon become obsolete. For instance, the popular genome-engineering tool, CRISPR, ...mouse lines, such as BAC transgenic or traditional knockins/knockouts, a thing of the past. In the sequencing...

...bioinformatic and proteomic tools to discover genes and proteins associated with pathogenesis. In his future work... T7 polymerase, EcoRI, and various fluorescent proteins. Dr. Duedu’s plans for these plasmids have a heavy...PCR. We hope these plasmids will help Dr. Duedu inspire new Ghanaian scientists to continue to do excellent...have received equipment through Seeding Labs’ Instrumental Access program. We are excited to discover what...Apply to Recieve Equipment through Seeding Labs' Instrumental Access Program Additional Resources on the ...

...does all of its damage with just seven structural proteins. But, as NPR explained, it’s not the virus itself... critical for viral attachment and fusion. Once inside a host cell, new evidence in PLoS Pathogens shows...shows that the Ebola virus glycoprotein remains essential in allowing those infected cells to go undetected...in a novel way, by shielding host cell surface proteins. Researchers at the University of Virginia School...California, Santa Cruz, offers sequence data on five strains of Ebola representing the latest outbreak and earlier...

...researchers led by Dr. Jorge Ruas of the Karolinska Institutet, the discovery really wasn’t what they’d...those mild stressors as usual, but somehow their brains were protected. Effects of Activation of the PGC... Moon and Henriette van Praag of the National Institute on Aging in a commentary about the new work published...explore. “It will be important to identify the key proteins and metabolites that are dynamically secreted ...

...covalent modifications to DNA or histones, the proteins that help package DNA, were associated with gene...With the help of guide RNAs, fusions between Cas proteins and the epigenetic modifier could be targeted ...cytosine demethylation of DNA. However, several proteins in the DNA oxidation and repair pathways work ...of the larger pMVP/pMAGIC cloning system, which contains Cas9 fusions for several epigenetic modifiers,...Sander JD, Reyon D, Bernstein BE, Costello JF, Wilkinson MF, Joung JK (2013) Targeted DNA demethylation...endogenous genes using programmable TALE-TET1 fusion proteins. Nature Biotechnology 31:1137–1142 . https://doi.org...

...immune systems that provide bacteria protection against infectious agents. Several enzymes behind this ... from the Department of Energy’s Joint Genome Institute for uncharacterized genes that were proximal to...40-70 kDa), which is half the size of other Cas proteins found in the so-called class 2 CRISPR-Cas systems... of the larger and more complex Cas9 and Cas12 proteins. Cas14 cleaves ssDNA But what is the function ...providing a gRNA specific of a viral sequences, for instance, the enzymes are activated and can cleave fluorescent...

...for this tool? Howarth: Polyproteins are useful for mechanical study of proteins by atomic force microscopy...SpyLigase can be used to link affibodies or antibodies against common tumor markers to subsequently capture cancerous...SpyTag/SpyCatcher system, to be able to grab on to proteins irreversibly in a genetically encoded way. SpyTag... bacteria have this chemistry in their surface proteins. It is best studied in the pili that they use ...

...pooled libraries include enrichment against kinases, cell cycle proteins, and more.  “In the past, genome... really not possible before,” said the Broad Institute's Ophir Shalem. “Whole regions of the genome which...delivery vehicle, Neville Sanjana, also of the Broad Institute, said they were able to produce a very diverse... of one of the Science papers, told the Broad Institute. “This will greatly aid efforts to understand ...

...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!    ...

...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...

...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...

... 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...

...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–...

... 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...

...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...

... thanks to our guest blogger Rachel Rubinstein! Rachel Rubinstein is currently a Field-based Product Manager...post was contributed by guest blogger, Rachel Rubinstein, a field-based software product manager at Oxford...your network to your advantage  As the profession gains more traction in the sciences, I believe that more...customer in mind. You can follow her on twitter @rgrubinstein. Additional resources on the Addgene blog Discover...

...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...

...readouts for levels of different chemicals, hormones, proteins, etc., that you are interested in. The most obvious...could be a microscope, plate reader, or any other instrument which measured GFP levels and converted that ...includes tension monitors associated with specific proteins of interest such as actinin. When tension is high....2019.00474 Resources on Addgene.org Fluorescent Proteins: Biosensors Viral Service: Biosensor AAV Viral...Epigenetics with a FRET Based Biosensor Fluorescent Proteins 101: Fluorescent Biosensors ...

... may feel it’s overwhelming or challenging. So instead, let’s focus on practicing with a short and accessible...information? This can be physical or digital. For instance, you would present information differently in ...image on a protocol page on our website, as an instructive step-by-step guide.  Finally, when writing your...after centrifugation and the syringe needle is inserted bevel-up at the interface between the 40% and ...captured at the 40% layer. To collect purified AAV, insert a syringe at the interface of the 40% layer containing...

...comprised of two tandem fluorescent proteins: a relatively pH-insensitive RFP variant, DsRed.T3 and a pH-sensitive...the degradation rate of radio-labeled long-lived proteins as a proxy for autophagy. Cells are cultured with...washout period removes radio-labeled short-lived proteins primarily via proteasome degradation. Autophagy... to giving more fine-grained detail about what proteins or organelles are being degraded) and it is slow...

...discussions.  Pro tip: Instead of connecting with someone you don’t really know, follow instead of connecting....information available. When I am speaking at a new institution, for example, I will often see a whole bunch ... “a postdoc from this Uni viewed your profile” instead of “Joanne Kamens, PhD viewed your profile.”  The...option for sending LinkedIn messages to Click Send instead of Press Enter to Send. That way you can type a...

...postdoctoral researcher at the Noble Research Institute, co-instructed the ASPB workshop and authored a three-part...well received, and then when the team started brainstorming ideas for the next workshop, they realized that...in the group was able to make it physically to instruct the workshop at the conference, but because each...of the scientific community. Lessons learned Instead of continuously talking about a problem and potential...

...resources. There are three types of cognitive load: intrinsic cognitive load is the effort associated with a...show frustration if they're incorrect; take it instead as valuable feedback.  Listen. Think about the ...strategy. Don’t assume it’s the audience’s fault; instead look for ways to improve how you're communicating...If something I was communicating didn’t click, instead of blaming the audience, I would think about how... wasn't. Maybe I needed to employ a visual aid instead of describing something verbally. Sometimes a group...

...for every assay. Antibodies that are generated against a linear antigen tend to work very well for applications...application. Take immunohistochemistry (IHC), for instance. While an antibody may be validated for IHC, not...region highly conserved across species? If the proteins are highly homologous between your target species... each isotype or species to easily discern the proteins in your assay.  Narrowing the field After working...compare the staining patterns of multiple antibodies against the same target; tagged protein strategies that...