We narrowed to 814 results for: Ran;

...proteins needed for the virus to replicate,  and the transfer plasmid which contains the viral genome. In today...and cap are expressed from a separate plasmid in trans, freeing up space in the viral genome so that a ...Replication initiates from the AAV genome ITRs on the transfer plasmid, which contain regions of dsDNA. To generate...capsid. Rep proteins are also involved in the transcriptional regulation of the AAV p40 promoter which drives...promoter. Alternative splicing and differing translational start sites generate the three VP proteins. ...Assembly-Activating Protein (AAP) and the recently discovered Membrane Associated Accessory Protein (MAAP) from an alternative...out this blog post to learn more about the AAV transfer plasmid! References: Naso MF, Tomkowicz B, ...

...products are then transformed directly into chemically competent E. coli and transformants selected with ... for further purification before transformation. After transforming cells, you should perform colony ...consisting of digestion, purification, ligation, and transformation. While restriction cloning is still routinely...+ + Gel Purification + - Ligation + - Transformation + + The FastCloning process in brief In ...for this procedure to work properly. Directly transform chemically competent cells with the digested mixture...-cycle PCR leads to the most colonies after transformation. When a 1:1 vector:insert ratio is used in ... and 2 µL of digest product are used in the transformation, >70% of the colonies are correct. Good luck...

...vaccines differed depending on the variants (Garcia-Beltran et al., 2021). Antibodies from individuals who ...and B.1.351 variants tested. Image from Garcia-Beltran et al., 2021 with permission.   The spike variant...represent lineages and variants that are actually transmitting out in the world” says Balazs. “By combining...quantified in terms of luminescence. Image from Garcia-Beltran et al., 2021 with permission.   Mutations ...with various pseudotyped virus. Image from Garcia-Beltran et al., 2021 with permission.   Difference ...https://doi.org/10.1038/s41586-021-03471-w Garcia-Beltran WF, Lam EC, Astudillo MG, Yang D, Miller TE, Feldman...Nitido AD, Murali MR, Alter G, Charles RC, Dighe A, Branda JA, Lennerz JK, Lingwood D, Schmidt AG, Iafrate...

...from one that represses transcription to one that activates transcription.However, this system lacks spatio-temporal...barbara_synbio) May 13, 2021 The original AraC transcriptional regulator Their system is based on the AraC...AraC protein that controls transcription from the PBAD promoter. “AraC is widely used in synthetic biology...It relies on the sugar arabinose to activate transcription by causing conformation changes in the AraC ...domains of the BLADE dimerize and activates transcription. The team created two constructs, among others...your gene of interest, all you have to do is co-transform pBLADEONLY. That way, a previously cloned gene...with AraC,” says Di Ventura. BLADE activates transcription with the flick of a light switch and shuts off...

...one of the most inclusive databases of post-translational modifications, identifies a staggering ~250,... enabled researchers to create an orthogonal translation system (OTS) for the incorporation of phosphoserine...system defined three components required for translation (Figure 1): A tRNA that recognizes a specific...elongation factor that allows proper extension and translation of the pSer-incorporated polypeptide. These ...Sep-OTS, contains the components required for the translation of pSer-incorporated proteins on a single plasmid...factor that accepts Sep-charged tRNAs during translation. Many ‘flavors’ of Sep-OTS plasmids are available...bacterial strain would result in read-through translation of many stop codons and detrimental effects on...

...remote collaborations Transferable Skills Guide: External collaborations Transferable Skills Guide: Teamwork...to explore this role, how to seek out such an arrangement, and how to operate once in that position. What... plan as well. Making logistical and funding arrangements Visiting scientist positions take commitment...structured visiting program, you will have to make arrangements on your own. This will include, but is not limited... limited to temporary housing, transportation, living expenses, and necessary reagents. Your primary lab...these expenses, or you may need to seek out travel grants available from third parties or your home institute...generate data that will contribute to a publication? A grant? A patent? It is never too early to discuss authorship...

... Wenning Qin. Single-stranded DNA repair templates Short single-stranded DNA templates have been shown...perform in vitro transcription, and use the IVT product as a template for reverse transcription to create your...order to insert two loxP sites. Since two double-strand breaks are made, it is common to see deletions ...traditionally been difficult to produce long single-stranded DNA, but Miura et al. (2015) developed the ivTRT...structure. Today, multiple companies offer long single-stranded DNA production services or kits, including the...microinjection and electroporation methods using in mouse transgenics. Designing an Easi-CRISPR knock-in allele To ...used guides targeting the same or complementary strands, as well as guides facing towards or away from ...

... from Abhi Aggarwal.   Brandán Pedre Pérez: Redox biosensors Brandán Pedre Pérez is a postdoc at... enable multiplexed imaging of neurotransmitter, calcium, and membrane voltage,” says Tian. Figure...Congratulations to the five winners! Abhi Aggarwal Brandán Pedre Pérez Elizabeth Hubbard Haoyi Qiu He Tian...hippocampal cultured neurons, imaged 14 days post-transfection, excited at 505 nm (right). Scalebar represents...hydropersulfide-propagating enzymes 3-mercaptopyruvate sulfurtransferase (MPST) which catalyzes the desulfuration of...an enzyme-bound hydropersulfide that is then transferred to thiol-containing acceptors. These hydropersulfides...redox modification, the MPST hydrosulfide can be transferred to the roGFP2 cysteine thiols and change its ...

...the co-transduction by two viruses would lower editing efficiency, the split editors transduced at levels...capacity of 4-5 kb limits the delivery of larger transgenes, such as base editors which have a gene size ...base editor is split into two plasmids and co-transfected into cells for reconstitution. Image from Levy...-opsin GEVIs. With an increase in the neuron’s membrane potential, Ace absorbance increases and results...promoters, CRISPRi parts, selectable markers, transformation acceptor vectors, and sequences for genomic...plasmids using the Cyanogate Kit and subsequent transformation into cyanobacteria. Image from Vasudevan et... evaluated CreLite for cell tracing using the transgenic zebrafish model, ubi:zebrabow. In this system...

...are not always easy to track at the transcriptional or translational level. However, several genetic tools...slightly slower scale.  All living cells with a membrane possess an electrical state. This state, known...is maintained by ion channels and pumps in the membrane. An emerging interdisciplinary field of biology...features increased brightness and improved plasma membrane specificity. Stable Step Function Opsin - A stabilized...Use of genetically encoded, light-gated ion translocators to control tumorigenesis." Oncotarget 7.15 (...PMID: 29684787. Oviedo, Néstor J., et al. "Long-range neural and gap junction protein-mediated cues control...PMCID: PMC2823934. Pai, Vaibhav P., et al. "Transmembrane voltage potential controls embryonic eye patterning...

...StayGold variants AAV.CAP-Mac for systemic gene transfer in primate brains Endothelial-specific AAV delivery...electroporation or poor cellular entry with other transfection methods. The Junwei Shi Lab addressed this problem...variants:   Figure 2: Inner mitochondrial membranes imaged with structured illumination microscopy...2023). StayGold variants for molecular fusion and membrane-targeting applications. Nature Methods, 1–9. https...breaching the blood-brain barrier and delivering a transgene, with neuron bias observed in young macaques and... viral vectors for functional intravenous gene transfer throughout the non-human primate brain. Nature...the Gradinaru lab, AAV1-X1, was developed by transferring the 7-mer peptide region from variable region...

... integration of a novel gene randomly into their genome, some transgenic strains are difficult to breed...expression level of the transgene. In existing strains of mice, the transgene can be maintained on a hemizygous...are many types of genetically engineered mice: transgenic mice, knockin and knockout mice, and conditional...that only one chromosome needs to contain the transgene in order to see an effect. Hemizygous mice can...breed. Some problems with transgenic mice that can impact reproduction include embryonic lethality, a short...expression level, and site of integration of the transgene. These problems can be mitigated in different ...different ways. Prior to engineering a transgenic strain, the promoter being used should be tested in cell lines...

...AAAS Science and Technology Policy Fellow at the Veterans Health Administration. “But, seriously, what even...Science and Technology Policy Fellow (STPF) at the Veterans Health Administration, I could never get one straight...variety of backgrounds with technical expertise that ranges from astronomy to bioethics to chemical engineering...happen at the national level within the executive branch or in Congress, or can also happen at the state... early career researchers who have decided to transition out of academia from graduate school or a postdoc... came to DC to do either a sabbatical year or transition away from academia as tenured professors. There...taken through your own personal lens.  For me, the Veterans Health Administration was the perfect position...

...vivo starts with transducing cells with the CRISPR library in vitro before transplanting them into mice....time. This reduces the chance of integration at random locations that could cause deleterious effects....portions of the AAV genome can integrate at double-stranded break points caused by CRISPR-Cas9 editing. The...The good news is that this integration is not random, nor is it a global occurrence, as integration sites...target cells or tissues. AAV also shows improved transduction efficiency over lentivirus in many mouse tissues...019-12449-2 Pupo, A., Fernández, A., Low, S. H., François, A., Suárez-Amarán, L., & Samulski, R. J. (2022..., U., Jabaudon, D., & Platt, R. J. (2023). Transcriptional linkage analysis with in vivo AAV-Perturb-seq...

...only have to worry about cloning your transfer plasmid. The transfer plasmids have different packaging capacities...AAV and lentivirus requires three plasmids — a transfer plasmid containing your gene of interest plus ...in mammalian cells is relatively quick. After transfection, HEK-293T cells start producing virus within...systems, it is standard to do only one passage (transfection). This differs from typical insect cell preparations...an additional couple of days, as it requires transduction of more cells, while AAV can be titrated following... baculovirus requires only two plasmids — your transfer plasmid and the baculovirus construct, called ...between insect and mammalian systems. Baculovirus transfer plasmids have a much larger packaging capacity...

...tool that responds strongly to magnetic fields (Frank Hayward et al., 2024). They deposited their plasmid...proteins. Motivated by curiosity Although a wide range of fluorescent proteins and optogenetic tools exist...when a small magnet was passed over the sample (Frank Hayward et al., 2024). The fluorescence intensity...the magnetic field is on. Image reproduced from Frank Hayward et al. 2024 under CC-BY license. They...mScarlet, and AsLOV2. After several rounds of semi-random mutagenesis and screening, the EGFP and mScarlet...the magnetic field is on. Image reproduced from Frank Hayward et al. 2024 under CC-BY license.   ...tools for biological applications across a wide range of topics. He embraces open science, self-publishing...

...separation of the two DNA strands.  Annealing- The temperature is then dropped to a range in which the forward...the primers can anneal to the single stranded DNA template strands. DNA polymerase is also stable at this...template DNA to amplify from and unique single stranded DNA primers that bind upstream (5’) and downstream... process as DNA polymerases require an existing strand of DNA to add nucleotides to.  Using these reagents...reaction:  Denaturation- To amplify DNA, the two strands of the template DNA first have to be separated....used to quantitate RNA levels using reverse transcription-qPCR (RT-qPCR). The first step of this process...requires RNA to be converted to cDNA using reverse transcription and the cDNA is subsequently quantified by qPCR...

...or Förster] resonance energy transfer). FRET occurs when energy transfer from one FP (e.g. CFP) excites...classified by the emission color (emission wavelength range) FPs are usually classified by emission color as...as outlined below (or emission wavelenght range).  By mutating GFP, the variants blue FP (BFP), cyan FP...nm Green 499 - 519 nm Yellow 524 - 538 nm Orange 559 - 572 nm Red 574 - 610 nm Far-Red 625 -... ≥ 670 nm In addition to emission wavelength range, there are other traits that need to be considered... green or red light. For example, T-Sapphire, LSSmOrange, and LSSmKate. Fluorescent Sensors: These FPs...related colors. Furthermore, most FPs  have a broad range of emission which will be detected by longer-wavelength...

...Uniform Biological Material Transfer Agreement (UBMTA) for its material transfers. The UBMTA was drafted by...organizations Assurance that recipient may not use the material for a commercial purpose Assurance that recipient... has time for paperwork? Here’s why a Material Transfer Agreement (MTA) matters: Science thrives on collaboration...universities and institutions require them for material transfers. When Addgene entered the plasmid sharing space...receiving institution. Therefore, many technology transfer officers are familiar with the UBMTA and are happy...  material transfer agreement...

... dilution should fall within the range of the assay for a wide range of titers. We usually dilute our ...determine if your titers are within the expected range. We have also found that generating the standard...For these reasons, we are in the process of transitioning our titering method from qPCR to ddPCR. Droplet...virus. It is important to note that the dynamic range of the ddPCR is between 1 and 100,000 genome copies...per reaction. Since AAV titers tend to be in the range of 1012 to 1013 GC/mL, you must serially dilute ...million.  After making the dilutions, they are transferred to a new plate containing the mastermix which...generator, you will have to be careful about transferring your droplets from the droplet generator to ...

...dCas9 has focused on transcriptional activation using a fusion to a transcriptional activator such as VP64...repression of transcription through binding a promoter region to inhibit association of transcriptional activators...locus of interest, the promoter region of the transcription factor IRF-1. Immunoprecipitation, using an ...2018). Furthermore, the Fujii Lab generated a transgenic mouse line and retroviral expression system for...Tapscott Lab identified regulators of DUX4, a transcriptional regulator that when inappropriately expressed...PMCID: PMC6001023. Fujita, Toshitsugu, et al. "Transgenic mouse lines expressing the 3x FLAG‐dC as9 protein...CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes." Cell 154.2 (2013): 442-451. ...

...false positive colonies. Transformation - the linear fragments are transformed into E. coli and plasmid...assembly of a circular plasmid in vitro, before transforming it into E. coli for propagation. However, while...using a simple two-step, 2 hr protocol prior to transformation (Figure 1). As this approach requires no special... encode homology arms to allow immediate co-transformation with linearized vector backbone. DpnI digestion...failed so far. What is most important is the transformation efficiency of the strain: when asking the cell...high fidelity DNA polymerase This will minimize random errors during PCR: we recommend Phusion or Q5. ...investigating the molecular biology of synaptic transmission and plasticity.     Javier García-Nafría is ...

... inducible translocation trap (ITT) system to analyze signal-induced nuclear translocation of proteins...functions such as epigenetic regulation and transcription by using locus-specific chromatin immunoprecipitation...biochemical analysis of genome functions such as transcription and epigenetic regulation. To elucidate molecular...Addgene realizes the One-Stop shopping for a wide range of plasmids. The merit of depositing plasmids to...considering the peer-review system of journals and grant applications, for example. However, at the same ...living for Japanese, which is not trivial, and the grant situation is currently much better than that in ...tough competition for academic positions and poor grant situation. So, young researchers need to publish...

...injections can be found here [5, 6]. Opsin transgenic mice Transgenic mouse lines have also been engineered...specific brain region can result in a particular neurotransmitter release or post-synaptic current. Fortunately...available, instructions for obtaining a material transfer agreement, and list of vendors (such as the vector...at a similar spectrum as HR and also results in membrane hyperpolarization but unlike HR, this is achieved...[10]. Unfortunately, opsins are not actively transported and must passively diffuse so, depending on the...axonal tracing with fluorescent proteins in nontransgenic and cre driver mice. Current protocols in neuroscience...et al. A toolbox of Cre-dependent optogenetic transgenic mice for light-induced activation and silencing...

...protease allows a transcription factor to enter the nucleus and modulate transcription of the mCitrine ...HeLa cells were transfected with plasmids encoding FPs tagged with the plasma membrane targetin sequence...non-specific labeling. For labeling the plasma membrane, they tested different target sequences for improved...expression can vary significantly depending on transgene design and delivery. To overcome these variations...into genomic DNA with the help of the piggyBa transposase. Image from Hazelbaker et al., 2020. The lab...activating expression of TCF4 while monitoring transcript and protein levels. Plasmids for multiplexed ... new serotype PHP.V1 that exhibits efficient transduction of vesicular brain cells. Find this AAV at ...

...amplification) or reverse transcriptase (RT)-RPA, respectively. RPA is coupled with T7 transcription to convert amplified... method termed DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR) (Chen et al., 2018). Both SHERLOCK...almost anywhere as lateral flow strips are easy to transport and work rapidly, providing reliable results in...    DETECTR- DNA Endonuclease Targeted CRISPR Trans Reporter Cas12a (Cpf1) is a CRISPR Cas variant that...discovered Cas12a’s ability to cleave non-specific (trans) ssDNA and harnessed this ability to create a DNA...DNase activity of Cas12a is initiated. Surrounding trans-ssDNA, including the ssDNA-FQ reporter are subsequently...Servellita V, Singh J, Miao X, Streithorst JA, Granados A, Sotomayor-Gonzalez A, Zorn K, Gopez A, Hsu ...

... after each transformation and/or use different E. coli strains with higher transformation efficiency....vector with two other independently regulated transcription units. Created with BioRender.com.   Planning...MoClo kit through their online catalog pages. We guarantee this will save you a lot of headaches down the...robot, you can set it up to automate the reagent transfer and the restriction–ligation reactions in a 96...colonies) after each consecutive assembly and transformation round, due to the decrease in ligation efficiency...’re cloning in high-throughput, we recommend transforming into chemically competent cells in a 96-well...have what look like positive colonies on your transformation plates. However, even though the MoClo system...

...promoter of another gene, thereby driving aberrant transcription. That being said, we generally pick guides...been significant work on constructing Cas9 transcriptional activators. These constructs allow for the ...Cas9 deficient in DNA cutting activity to a transcriptional activation domain (Fig 1). Two ways to use...picking whichever guides are closest to the transcription start site (TSS). We recommend targeting the...Latency Reversal Using CRISPR/Cas9-Derived Transcriptional Activator Systems. PLoS ONE 11:e0158294 . https...Church GM (2015) Highly efficient Cas9-mediated transcriptional programming. Nat Methods 12:326–328 . https...Nishimasu H, Nureki O, Zhang F (2014) Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex...

... cell types. Specific transgene expression can be also achieved in nontransgenic animals by using cis ...delivery by increasing transgene cargo capacity, enhancing anterograde transport efficiency, and engineering...proteins that determine the AAV serotype. During transduction, the viral capsid proteins bind to the target...serotype used for a specific cell type. Find some transduction characteristics of select AAV serotypes in Table... AAV vectors are common approaches to control transgene expression by restricting expression to genetically...enhancers, multicistronic vectors, and post-translational regulatory elements. Examples of these approaches...

...enzyme or transcriptional activation, conformational changes, translation, and protein translocation. Targeting...triggers CaM/M13 interaction that increases FRET transfer between the fluorescent proteins. FRET is highly...characteristics like pH, redox state, and voltage across membranes. These sensors generally follow the same pattern...an organelle-specific targeting signal or a transmembrane domain, biosensors can be targeted to a particular...endoplasmic reticulum (ER), vacuole or cellular membranes. Examples include blue flame mitochondria-targeted...

... the CAR facilitates signal transduction and release of cytotoxic granules from the T cell. When a CAR...., 2012, Bates and Power., 2019): rapid blood clearance, which is useful for imaging applications; better...small size (Bates and Power., 2019). Their rapid clearance from blood can be a drawback for therapeutic applications... has led to their application for basic and translational research.  Basic research applications of scFvs...single molecule tracking in living cells. The HA frankenbody is an HA tag detection probe. It contains an ...to a fluorescent protein such as GFP. The HA frankenbody works just like an antibody-based probe, but ...generate since it’s genetically encoded.   Translational applications of scFvs: Just like antibodies...

...ready-to-use viral preps   Improved cell-free transcription/translation system to examine biosynthetic pathways... Gabrielle Clouse Recently, cell-free transcription/translation systems (also referred to as TX-TL systems...modifications to edit DNA without requiring a double-stranded break. The Xiao Wang Lab has recently deposited.... These plasmids use “Cas9-mediated adenosine transient reporter for editing enrichment”, or XMAS-TREE...and enrichment use of XMAS-TREE. a) Cells are transfected with three different vectors and flow cytometry...identification of essential MYC binding sites in a wide range of human cells.  A new pooled library from Junjie...

...grateful to the grant officers at Fast Grants. We received a miraculous grant from Fast Grants, which was ...studying the coronavirus. Addgene’s Technology Transfer Team also moved fast to ensure that the majority...collaborations as well as provides visibility for grant-driven research being done in all labs sharing materials...materials via Addgene. Perhaps if each of many grantors supporting research could provide Addgene with... for supporting Addgene by providing a generous grant. This support will enable us to continue all operations...

...fluorescent protein (XFP) genes in a transgene. When multiple copies of the transgene cassette are introduced, stochasticity...fluorescent proteins is only possible at low copy number ranges, resulting in low fluorescent protein level. The...gene delivery is more common than conventional transgenic strategies. In such vector-based gene delivery...in the original method, we used the Tetracycline trans-activator system to boost the expression levels,...levels, and we named the system Tetbow (Tetracycline trans-activator Brainbow) (Sakaguchi et al., 2018) (Figure...expression level of XFPs, most likely by suppressing transcription. It is critical to express a minimal amount ...Bio-Protocol 8 (2018): e3046. Livet, Jean, et al. "Transgenic strategies for combinatorial expression of fluorescent...

... and not in academia is run by grant funding. Learning to write grants quickly, efficiently and effectively...apply to or offer to help with lab grant writing. You may not get the grant or your PI may edit everything...thinking about future career options. 5. Write a grant - even if you don’t need one A lot of science in...valuable skill. There are many different kinds of grants that you can apply for. If you are in a lab, your...Here are some sites that have compiled lists of grants: Science: Where to Search for Funding The Ohio...For Your Research University of Florida: Training Grants and Other Funding Opportunities L’oreal USA For...

...method is transfection of short interfering RNA (siRNA). Knockdown caused by siRNA is also transient, and ...interest. This approach often involves the transient transfection of expression plasmids in a given cell ...commercially available. Various proteomic and transcriptomic datasets can be used to select a candidate ...antibody should be able to detect even in cells transfected with low efficacy. But since there can be large...differences between endogenous expression and transfected overexpression, this approach cannot confirm ...workshops. Let’s use TIM-1 (Single pass type-1 membrane protein), which has been designated CD365 in the...the antibodies both recognized the target when transiently overexpressed in CHO cells and displayed similar...

...covalently joined, scarless DNA – perfect for transforma­­­tion or as a PCR template. Some of the applications...by using NEBuilder which requires only a single-stranded synthetic oligo to generate a single-guide RNA...RNA (sgRNA). Using only one single-stranded oligo, instead of the more typical two oligos, is more cost...annealing step usually used to create a double-stranded oligo. Generating an sgRNA construct becomes as...target region. Assembled plasmids can be directly transformed into E.coli for plating, screening and generation...complexity, is achieved with synthetic oligos with a randomized region between the regions of homology. Producing..., generating expression cassettes for yeast transformation can take 5 days. NEBuilder shortens that time...

...differently. Communication comes in many forms—from transparency about data to allow others to see what you actually...communication and sharing fits together to make for a transparent and collaborative research environment. Journals...Currently, most of the burden for pushing for more transparency in science communication falls on the shoulders...developed STAR Methods to improve the structure and transparency of methods sections, including clear information...researchers are not strongly rewarded for being transparent in their communication or making their data and... to a large extent, hold the keys to getting transparency and communication further up the collective ...management plans and for evidence of sharing for grant renewal—but there is clearly room to do more. If...

...2015, there were six of us. We were initially strangers but we all shared a passion for DIY Biology and...December 2015 we held our first intro to bacterial transformation course (read our blog post on it and to see...consisting of over twenty members and volunteers. Transforming citizens into scientists Anyone can become a...dedicated time at BosLab, he became our bacterial transformation guru and instructor for our intro to biohacking...biohacking courses. Our members pursue a diverse range of projects at BosLab. Some are entrepreneurs using...RNA with small molecules. Other members’ projects range from hacking probiotics to filming educational lab...BosLab. Once he optimizes plant cell culture and transgenesis he wants to share his knowledge with the community...

...Cas enzymes) cleaves both strands of its dsDNA target in cis and has no trans activity. AsCas12a, on the...cleaves ssDNA in trans. In sum, CasPEDIA documents the target substrate (DNA/RNA and strandedness) along with...Cas enzyme: Nuclease properties (CasID): Cis/trans activity, PAM requirement, gRNA, and multiplex ability...features: CasID CasPEDIA will display both the cis and trans activity of each entry. For example, SpyCas9a (one...can manually enter details to refine your search (trans activity, multiplex ability, gRNA design, etc.).....32942/X2C31F     Makarova, K. S., Wolf, Y. I., Iranzo, J., et al. (2019). Evolutionary classification...