...Deisseroth, K. (2005). Millisecond-timescale, genetically targeted optical control of neural activity. Nature Neuroscience...Ganguli, S., & Deisseroth, K. (2019). Cortical layer–specific critical dynamics triggering perception. Science... activated tools, can control everything from electrical activity to gene expression, and even CRISPR ...through multiple intra-cellular signaling cascades. Optical methods for controlling GPCR activation have been...stimulation. GPCR signaling GPCR signaling is a complicated process involving many downstream signaling mechanisms...break your experiments!  First, closely read the publication, including the supplemental data. You’re most... both of which have extensive collections of publications and information on hardware, optogenetics tools...

...target specific tissues, even when administered systemically, without causing disease? Recombinant adeno-...in-stock viral prep service.  AAV5 AAV5 is the most genetically distinct AAV. Though AAV5 is one of many serotypes...vessel barriers. It is especially popular for applications targeting liver tissue. In addition, it has ...the blood-brain barrier, making it useful for applications targeting the CNS (Issa et al., 2023). Additionally...al., 2019 Liver AAV8 Sands, 2011 Lung pUCmini-iCAP-AAV9.452sub.LUNG1 Goertsen et al., 2022 Pancreas...treatment, which can pose problems for gene therapy applications. Cross-reactivity of AAV serotypes varies by...on the AAV capsid. Molecular Therapy. Methods & Clinical Development, 26, 331–342. https://doi.org/10.1016...

...variety of research applications, including making transgenic cell lines and genetically-modified cell lines...facilitates the integration of the transposon specifically at ‘TTAA’ sites randomly dispersed in the genome...piggyBac™ technology can be used for numerous applications, including transgenesis, gene-trap screens, ...of interest. The piggyBac™ system allows for significantly enhanced random integration efficiencies that... If the cell’s chromatin structure undergoes significant rearrangement (such as when a stem cell initiates...of the piggyBac™  transposase/transposon gene modification system and the scarless, Footprint-Free™ removal...re-integration from the piggyBac™ donor, which is typically found with the wild-type piggyBac™ transposase...

...degraders provide distinct advantages over pharmacological inhibitors including rapidly depleting a protein-of-interest...resistance to inhibitors, and inducing prolonged biological effects. While targeted degradation of endogenous...here. The Methods section of Nabet et al., Nature Chemical Biology additionally provides detailed information...FKBP12F36V binding ligand (ortho-AP1867) via various chemical linkers. Our current lead dTAG molecule is dTAG...FKBP12F36V-tagged LACZ) with dTAG molecules in your biological assay of choice to ensure that there are no toxicities...µM (see Figures 3 and 5 in Nabet et al., Nature Chemical Biology for examples). Monitor neo-substrates ...experiments (Nabet et al. and Weintraub et al.), chemical and genetic rescue experiments (Nabet et al.),...

...since other signaling networks can also be non-specifically activated and, like the game of telephone, often...must be transmitted by intermediate messengers. Optical control of kinase activity can provide greater ...greater spatiotemporal resolution than pharmacological or genetic approaches, but only a few such methods exist...Problem:  There is no common method for creating optically controlled kinases. The Solution: In Zhou et al...and MEK1 is active or “on.” Dronpa’s color thus indicates MEK1’s current state (Green when off, dark when...kinases good for? There are a number of potential applications for photoswitchable kinases.  Below are a few...endogenous ERK, and its localization serves as an indicator of MEK1 activity. When MEK1 is active, it phosphorylates...

...pathways and how they lead to neurological phenotypes observed.” Using genetically-encoded biosensors and iPSC-derived...Scientific Advisor. It’s possible that solute channels typically expressed on other cell types can compensate for.... “This proof of concept study will help in identification of new targets that could greatly enhance our...understand so that we could possibly develop pharmaceutical interventions that would inhibit IL-36 release...genes from the X chromosome are expressed at significantly reduced levels in women with TS (Trolle et al...Terry Hébert, a professor and assistant dean of biomedical science education at McGill University.  These...58 individuals with GNB1 disorder since the identification of this disease in 2016. The lab aims to “expand...

...and can be specifically targeted to a number of different cell types even when systemically administered... of AAVs contain two numerical designations, separated by a slash, that indicate the ITR and serotype,...experiments. Recombinant AAVs (rAAVs) The primary application of AAV is delivery of genetic cargo to a host...with maximum “trunk” space for genetic cargo. Typically, when researchers speak about AAVs, they are actually...transduced gene is episomal and does not divide and replicate in the same way the host DNA does. Because of ... since you don’t have to wait for the host to replicate the DNA before expression. Now that you’ve got... S., et al. “Various AAV Serotypes and Their Applications in Gene Therapy: An Overview.” Cells, 12(5),...

... antibodies may be indicated on occasion, usually after other signal amplification techniques have been...IgG2 or IgG4. This can be important for some applications. Assay use While secondary antibodies are useful..., the flexibility of their use varies across applications. Western blots, for instance, almost always ...For the latter three, secondary antibodies are indicated when the protein of interest is found in low amounts...been ruled out.  Ease of access Signal amplification isn’t the only benefit of secondary antibodies. Finding...which means that indirect methods are often significantly longer than direct ones.  Even though the secondary...quantitative analysis of a sample. Instead, relative quantification can be done using either the direct or the ...

...from the serum. Protein A/G purification Protein A or Protein G purification is one common method. These... the desired level of antibodies is reached - typically within 2-4 months. After the animal has the desired...reagent that works for your species. Affinity purification Only up to 5% of the IgG antibodies produced...specific for the immunogen. A different type of purification can be performed to isolate antibodies only ...inexpensive. Their short production time and the bulk purification steps keep the cost low compared to other antibody...are particularly useful for certain research applications. Many secondary antibodies are polyclonal, as...target protein thereby greatly increasing signal amplification.  When to stay away from polyclonal antibodies...

...propagated it, separated it into roughly identical replicate groups, and treated these groups with the...among the separate replicates would have different labels - i.e. they would stochastically arise from different...summarized in a recent Nature Medicine paper. In the publication, Bhang and coworkers used the ClonTracer library...below): 1. Resistant cells among the separate replicates would contain the same labels - i.e. they would... Looking at barcode abundances among multiple replicate populations, they found that similar barcodes ...populations were shared by at least one other replicate; prior to treatment, there are small (previously...such that initial resistant populations can be eradicated up-front quickly. This approach is in contrast...

...such as affinity purification mass spectrometry (AP-MS), lack the ability to specifically identify proteins...the validation of a binary PPI as well as the identification of additional interacting factors. Problems...detect weak and/or transient PPIs. Many PPI identification techniques rely on protein interactors to stay... AP-MS. This requirement often prevents the identification of weak and/or transient interacting proteins...lysed before the bait protein is pulled down, typically with an antibody. This can artificially select...controllable version of the BioID protocol. The critical component for both BioID and Split-BioID is BirA...Further characterization of these candidates is critical to understanding how they interact with the bait...

...past decade, primary cells remain difficult to genetically edit due to toxicity from electroporation or ...some cases) and minimal toxicity, including in clinically-relevant lines such as CAR-T cells. Plus, this..., its dimeric nature can interfere with some applications. Several labs have been working to produce monomeric... for molecular fusion and membrane-targeting applications. Nature Methods, 1–9. https://doi.org/10.1038...library generation and screening that led to identification and characterization of AAV.CAP-Mac. Image ...endothelial cell-specific tropism, with slightly reduced efficacy compared to AA9-X1.1. Importantly, AAV1-X1 is ...rodents and broad tropism in primates. Nature Communications, 14(1), 3345. https://doi.org/10.1038/s41467...

... utilize an array of tools that rely on DNA amplification and manipulation to produce and edit plasmids...plasmids. These are prone to error — both human and biological. Even the most high-fidelity polymerases can ...Potential points where DNA errors can be introduced, indicated by exclamation marks. Error points include places...places where DNA is being created, such as PCR amplification or bacterial growth. Created with BioRender....the correct inserts, tags, promoters, and other critical functional components. Fun fact! Some older plasmids...sequences. These sequences can also be confirmed or theoretical. No matter the level of information that is provided...elements, and nucleotide indels or mismatches in significant plasmid elements. Which differences are considered...

... series.  “The only healthy communication style is assertive communication.” - Jim Rohn, Author, Entrepreneur...Assertiveness? An assertive person is able to communicate their ideas confidently while also considering...of the most frequent sources of conflict is miscommunication. For example, you might misunderstand your...the wrong direction. The best way to avoid miscommunication is to follow up after every meeting with an...workplace, these strategies will help you to communicate with confidence earn respect from your supervisor...in an email, there is a high likelihood of miscommunication, which can lead to even more conflicts. If...manager, you can resolve most conflicts if you communicate your ideas assertively, listen to his or her ...

...lines of communication Few people actually like meetings, but run well, meetings can be a critical part of...depositors; Gizela, the lab member who knows how the physical kits are made and shipped; Chiara, the tech transfer...transfer expert who understands the somewhat complicated Material Transfer Agreement process that kits...repository, performing quality control, and providing technical customer service. Team members are highly trained...But this is a blog post, so here are just a few critical features based on my own experience: Leadership...should be on the team. As a team leader, I’ve (diplomatically) booted people off teams. I’ve also kicked ...are helpful, they are certainly not the only communication options. Addgene, along with an increasing number...

...the cell type under study. Many AAVs will show significant expression in a variety of cell types, but you...previous literature using AAVs for DREADD delivery indicates ~7-21 days are required for the onset of gene ...rate limiting step and can immediately undergo replication and transcription. However, with scAAV the effective...by the use of AAVs? 1) Combined functional and anatomical mapping of circuits In our lab, we use AAV-DREADD...these complexes are so robustly expressed that anatomical assessments can be easily performed too. While...identified this feature of AAVs as a new tool for neuroanatomical tracing (11). With the ability to produce neuron...Retrograde AAV Preps at Addgene 2) Comparison of genetically engineered receptors (i.e. chemogenetics vs. ...

... difficult to eradicate. You can suppress the growth of mycoplasma but cannot eradicate it completely....Contamination! Tissue culture media often have indicators included in the media that turns yellow from ...contamination Detection of mycoplasma by naked eyes or optical microscope can be very difficult, so how do you...cells detect mycoplasmas, they trigger a series of chemical reactions and this induces a noticeable color ...contamination, before using the cell lines for assay or analytical purposes. Use 0.1-micron filters as are they ...Fumigate the laminar airflow and the lab facility periodically. The CO2 incubators, used for maintaining cell...cleaned regularly and checked for contamination periodically. For more tips, check out Addgene's video Getting...

...most transposable elements (TEs) results in the duplication of this target site sequence at the site of insertion...This is similar to how retroviruses, like HIV, replicate. Class I TEs do not encode a transposase enzyme...enzyme.  Class I TEs are considered replicative since every time they jump they make a copy of themselves...retroviruses both in their structure and mechanism of replication. They contain two genes: gag and pol. The pol...they move. Most Class II transposons have a non-replicative “cut and paste” mechanism of transposition: they... it integrates, which produces a target site duplication (TSD). Created in BioRender.com.   Autonomous...transposition activity  Tol2 transposon plasmids    Applications of transposons Now that you’ve learned about...

...Sontheimer EJ, Mello CC (2018) 5′ Modifications Improve Potency and Efficacy of DNA Donors for Precision Genome...repair of a temperature-sensitive pha-1 mutation significantly enriches for knock-in of 2x and 3xFLAG epitopes...Mello lab also reported that modifying the 5’ modification of oligos used to generate linear dsDNA repair...melting and rapidly cooling dsDNA repair templates dramatically boosted editing efficiency (Ghanta et al., 2020... more efficient. Pure PCR repair templates are critical for efficient knock-ins (Ghanta et al., 2020)....Farboud et al., 2019). Work from the Mello lab has dramatically improved RNP editing with linear repair templates...Efficient Marker-Free Recovery of Custom Genetic Modifications with CRISPR/Cas9 in Caenorhabditis elegans. ...

...one another, and a value of “1” indicating those markers are identical and completely indistinguishable... tumors, and moreso than other methods, the identification and isolation of rare cell types. In recent...relies on “unmixing.” Unmixing uses a complex mathematical algorithm and single stained reference controls...spectral flow experiment and panel, there are a few critical factors to keep in mind in order to get the best...autofluorescence across various tissue types varies, it is critical to include an unstained sample for each tissue...complexity index ranges from 0-1, with a value of “0” indicating those markers are completely distinct from one...samples. If you are using conjugated dyes, it is critical that your controls and your sample antibodies ...

... molecular biology-grade agarose containing physiological concentrations of salts and glucose. If we circled...diluted from 10X and containing both Hepes and bicarbonate) is brought to room temperature and placed in..., and making sure we always maintain a backup replicates of each clone in case of contamination. We do...fluorescent protein), fluorescence microscopy can theoretically be used to select specific wells from the 48...further analysis. To assess CRISPR/Cas9 knockout efficacy, we have used both Western blotting and PCR, depending...population of transfected clones. The technique is applicable to a wide variety of cell lines, from HEK to ... terms of growth rates, morphology or other physiological properties. An advantage of this technique is...

...in normal biological processes and disease states. While LITE plasmids have clear application to anyone...effectors (TALEs) as anchors for genome engineering applications was still very new. Now, Konermann and her colleagues.... All of this requires genes to be regulated dynamically.” To understand how that works in memory formation...they could package LITEs in viral vectors and genetically target them to specific cell populations. They... the brains of living mice both quickly and significantly. Genes under LITE control showed a rise in transcription...prove the causal role of specific epigenetic modifications in the genome,” Zhang said in MIT’s press release...with LITE? Reference: Silvana Konermann et al. "Optical control of mammalian endogenous transcription and...

...MCC develops and makes freely available technical and pedagogical resources, offers mentoring for instructors...best teachers and mentors pass on more than just technical knowledge, shaping their trainees’ very way of...citrate synthase and presented their findings at the American Society for Biochemistry and Molecular Biology...their research capabilities and laboratory and critical thinking skills (Knutson et al., 2010a,b). Classroom-based...implementation. Funded by the NSF (with Co-PIs Jessica Bell, Lisa Gentile, Ellis Bell, and Amy Springer...Einstein: A Life. Chapter 17, Einstein Discovers America. New York./  Callahan, K., Peterson, C., et al....biochemistry laboratory: Part I – guided inquiry – purification and characterization of a fusion protein: Histidine...

...candidate in the Sternberg Lab at Columbia University Medical Center. DNA transposons are ubiquitous genetic ... and have been adapted for a wide variety of applications - as mutagenesis tools for genetic studies (... The process also creates a 5-bp target-site duplication (TSD), a hallmark characteristic of the Tn7-family...a multi-spacer CRISPR array. Workflow and technical considerations Making insertions in bacterial ...into cells and plate overnight Screen colonies phenotypically or by PCR/sequencing While CRISPR-transposons...few important details. For transposons to be specifically recognized during transposition, genetic cargos...Leveraging a unique marriage between distinct biological elements, CRISPR-transposons excel at programmable...

...retroviruses ” (Cullen, 1992) because of their more complicated genomes. Here, we will mostly use the gamma-retroviruses...(Prussin et al., 2015), only a small fraction typically cause disease. The gamma-retroviruses have their...wild-type viral particle — and of the normal, replicative life cycle — can help you understand these seemingly...of a non-living viral particle, and how we as dedicated researchers can exploit it. This blog was written...Hutcheson for her assistance with lentiviral replication.  Resources and References More resources on...101: Types of Viruses Viral Vectors 101: Viral Applications Resources on addgene.org Addgene's Lentiviral... proteins encoded by complex retroviruses. Microbiological Reviews, 56(3), 375–394. https://doi.org/10.1128...

...? The available filtering options now change dynamically based on the type of material and specific search...articles. From paper to plasmid Authors regularly indicate that their plasmids are available from Addgene...papers. Maybe they started their deposit prior to publication and were able to include specific plasmid ID ...terms we can use to find materials from this publication. Searching for the title of a paper works You...filters Not every plasmid search will start from a publication. You may have an idea for a plasmid that would...guide RNA / shRNA inserts Resistance Marker: This indicates the selectable marker used in your model system...Requests: Only see plasmids with more than the indicated number of requests Availability to Industry: Restrict...

... can often be accounted for using appropriate replicates and controls (see Tips below), and by validating...two-hybrid screening Minimizing false positives: Run replicates of the experiment to reduce the likelihood of...expression and correct folding, post-translational modification, protein degradation, access to the nucleus ...organisms, including bacteria, alternative fungi (C. albicans, pC2HB) (7) and mammalian cells. If the problem...co-expression of the enzyme responsible for the modification in the assay host strain can help. In order...that the configuration of your fusion proteins physically blocks the binding sites for the protein partner...Tachihara, K., Umezawa, Y.l. Split luciferase as an optical probe for detecting protein-protein interactions...

... at Sanofi Pharmaceuticals. This post highlights a rare disease, Fibrodysplasia ossificans progressiva...surpassed 60,000 views for the month of September! Historically we do better in September than in the summer...or you can’t quite remember the hazards of that chemical in your obscure growth medium, these apps can ...Binding Proteins," was contributed by guest blogger Jessica Sacher from the University of Alberta and describes...the post for the full details and many thanks to Jessica for all of her hard work and attention to detail...can find the rest of our September posts in chronological order here: Four Things I Learned at the 10... a Lab and a Project That’s it for now. As I indicated in the opening paragraph, I’m currently on my ...

...cancer. These later stages of a cancer diagnosis indicate, for Stage 3, that the cancer is large in size...size and may have spread while Stage 4 indicates that the cancer has spread from where it originated to ...asbestiform minerals such as erionite, are the only scientifically proven causes of the disease. There is no question... question on this point within the medical and scientific communities, and the link between asbestos and.... New techniques are currently being tested in clinical trials to detect mesothelioma earlier than ever...earlier, thus improving the patient’s prognosis significantly, but also to monitor those who may be at greater... working to fight the cancer.  Beyond early modifications to standard treatments and early detection, ...

...are subject to excision and ssDNA cleavage by canonical base excision repair enzymes. At the same time...2019). TREE is a real-time, fluorescent based identification system for the isolation of base-edited cell...targeted by a cytidine deaminase base DNA editor. Specifically the BFP mutant (BFPH66) contains a histidine...GFP positive cells isolated with TREE have a significantly higher frequency of genomic base pair edits ...GFP positive cells can be sorted and have a significantly higher frequency of base edits of interest than...robust induction of GFP fluorescence confirming the efficacy of GO as a base-editing reporter. As the initiation...markers, and other enzymes such as Cre-recombinase drastically expanding the base-editing reporter toolbox. ...

...were less significant than when non-targeting guides are used as controls. Less significant p-values lead... approach for identifying genes involved in a biological process. These screens provide an alternative...highly effective, are affected by low on-target efficacy, non-specific toxicity, and off-target effects...calling a gene a hit when really it’s not). In practical terms, this means that using safe-targeting guides...guides and may more accurately determine the significance of hits in non-growth screens. Off-target cutting...of on-target activity. However, there was no significant difference in depletion of ricin sensitizer genes...and do not have a major reduction in on-target efficacy. Results from Morgens et al. show that genome-...

...Addgene ID number, your publication will be listed on our website as a “Publication Citing this Plasmid” ...upon other researchers being able to find their publications in the first place. As a curator of the thousands...make it easier for you to find the plasmids and publications relevant to your research interests. We do this... this by promoting citation, supporting sophisticated search, and curating plasmid collections. Addgene's...number as well as the reference for its original publication. To help facilitate this process, we explain ...fruits of your labor and won’t have to repeat the modification. This will save both time and money. A quick...whether the plasmid has been used in your system. Sophisticated search You can search for specific plasmids ...

...any DNA editing functionality Collecting preclinical and clinical data about a disease or therapeutic Side-by-side... ReFigure. Reading and exploration including replications and experiments resulting in “negative data”...small selection of data found in full-length publications. As early career researchers, we wondered how...performance as in this antibody ReFigure Analyzing replications as in this ReFigure about whether Natronobacterium...reagent performs well, or you might publish a replication experiment that you conducted as part of setting...papers as they are not included in traditional publications. By submitting your data to a repository (Figshare...-registration, preprints, data, software, micropublications) in grant proposals. ReFigures are one such...

...If you use a kit for DNA purification or if you use a DIY purification protocol, you might have noticed... mutations in single stranded DNA undergoing replication.   DNase on the other hand can digest and degrade...degradation during plasmid DNA extraction and purification from E. coli is the presence of the protein ...most common E. coli strains used for plasmid amplification are endA negative, meaning the gene has been...chelating agents that can be introduced in the purification protocol to remove endonucleases activity. For...directly use your DNA preparation in any downstream application involving enzymatic reactions, a valid alternative...temperature for a short period, and there are indications that Tris buffer is better than water in these...

...The biomedical field is often concerned with understanding the cause of diseases and how to treat those...optimization is performed to take the drug to the pre-clinical and human trial phases.   So how does CRISPR factor...put into them in those later stages. Target identification Not all diseases are fully characterized or...simple pathology. In these cases, preliminary identification of potential targets by CRISPR can accelerate...enables researchers to select and generate physiologically relevant models. In complex diseases, it can...patient mutations which are variants of unknown significance – mutations with unclear effects on disease ...standard is to test the drug on models which are genetically proficient and deficient for the target. CRISPR...

...gene at once. Therefore, applications that require simultaneous modification of all alleles of a gene,...frequency three orders of magnitude higher than identical targeting vectors delivered through transfection... recommends designing homology arms with the modification of interest, ordering them from a gene synthesis...with a homologous repair template. It is this application - the creation of sequence-specific knockins ...clones, making it unsuitable for most in vivo applications.  Third, even in the most optimized tissue-culture...tissue culture based approaches that require modification of only a single allele of a gene, such as the...efficiently deliver large repair templates for applications that require large gene fusions. The current...

...where you can start to identify which items aren’t critical to your research success. If you can get away ... most start-up P.I.’s. It doesn’t have to be complicated, or difficult to create this list. Start BEFORE...resources to keep, and which ones to abandon. It’s critical to make these decisions in the following order... budget can help you compare the monetary and empirical value that helps you determine whether or not ...know how to decide which components are the most critical to your research, negotiations can be much easier...negotiating finances, instead of solely discussing numerical values, try to keep conversations focused on research...discounts, collaborations, access to internal award applications, etc. If this is the institute that can help...

...tolerates occasional mismatches or indels. Empirical tests indicated that a reasonable rule is to require at... While there were software tools available to biomedical researchers manipulating DNA sequences on a daily...features such as antibiotic resistance markers and replication origins, but there is extensive heterogeneity...different microbial strains. It proved to be impractical to catalog every variant of a feature. Instead...that have different properties. To prevent misidentification, we augment the database with new fluorescent...protein variants. The future of DNA feature identification What’s next for common features? We plan to...Genetics and Cell Biology at the University of Chicago. He was an early depositor at Addgene, and conceived...

...Storytelling is a crucial part of interpersonal communication, which makes honing your ability to tell stories...calmed the situation down by opening a dialogue specifically about the project and contributions, rather ...wherever it makes sense to start and try to be as chronological as possible — if you find yourself jumping backwards.... Your story should be too. Use drama Use any rhetorical technique you have in your arsenal to make sure...start point and it needs a good end point. One thematically pleasing option is to end at the present — ‘...other options. Choose something with emotional significance that is likely to be memorable. Work out what...Addgene Blog Check out our blog posts on science communication Find interviews scientists in SciComm careers...

...structural characterization and applications in fluorescence imaging." Biochemical Journal 400.3 (2006): 531...fluorescent protein: identification of the molecular determinants of color and applications in fluorescence... be used as a fusion tag for a wide range of applications from traditional imaging to FRET and stochastic...492 nm, respectively), they gave this a teal classification rather than cyan and named the protein mTFP1...FRET reporters. mTFP1 is also insensitive to physiologically relevant changes in pH. Find mTFP1 plasmids...excitation maximum 399 nm) because it does not significantly excite at 400 nm while EGFP does. Find mWasabi...