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...which leads to the desired mutation and excision of the vector backbone. This step requires “counter-selection...amplicons upstream and downstream of the desired deletion directly into cut vector with excellent efficiency...scarless mutations, ones that don’t leave any undesired scars in the chromosome. These methods roughly... powerful, but they rely on exogenous enzymes, require multiple transformation and plasmid curing steps... recombination enzymes can also accomplish the desired genomic modification. This technique is frequently...mutations. Ultimately, successful allelic exchange requires two sequential homologous recombination events...events, but because these events are rare, each requires selection. These two steps are (Figure 1): The ...

...topics we covered in this post. Besides direct labeling, indirect labeling can be used when suitable fluorophore-conjugated...label, where fluorophore-conjugated antibodies directly bind to epitopes of interest that are found in...through the cell membrane freely, while others require a permeabilized membrane to enter cells. Depending...and 'staining' to refer to small molecule dyes. Indirect labeling While antibodies used for target marker...western blotting or ELISAs.  A popular method for indirect labeling employs a primary epitope-targeting antibody...biotin plasmids at Addgene!     Figure 1: Indirect detection of target markers can be achieved through...Created using biorender.com. Another way of indirect labeling is through antibody host species reactivity...

...usage. Fast facts – PAM requirement: TTN or TTTN. Best for: homology-directed repair or therapeutic delivery...on choosing a Cas9 enzyme. Fast facts – PAM requirement: NGG and an expanding number of others. Best...proceed up to multiple kilobases, but only in one direction. Cas3 is thus not helpful for designer genome ...application potential as a result. Fast facts – PAM requirement: AAG or TTC  Best for: very large deletions. ...single strand DNA generation, such as homology directed-repair. Cas12a is also conducive for targeting...targeting AT-rich genomic loci as the PAM does not require any G’s or C’s. One curious feature of this Cas is...enzyme (about a third of the size) and does not require a tracrRNA (it can self-process pre-crRNA into ...

...lets you fire neurons with the flick of a light switch. Neurons are not typically persuaded to fire when ...and neurons don't fire. With blue light, ions flow into the neuron and causes it to fire. Created with BioRender.com...need high-intensity light for activation which requires implantation of fiber-optic cables into the brain...-throughput proteins engineering methods, like directed evolution, because channelrhodopsins are typically...them construct “designer” channelrhodopsins with desired characteristics. The model was trained to predict...and/or ChR2(H134R). Designer channelrhodopsins paired with a systemic AAV allow for minimally-invasive...express as many copies of the ChRs as compared to direct injection. This lower multiplicity of infection...

...are always required and lab coats are either recommended or required in BSL-1 and required in BSL-2 labs...Lentivirus (a type of retrovirus) and gamma retrovirus require more caution as they are integrating viruses – ...need to be handled in a BSL-2 (or BSL-2+) lab and require additional safety measures.   General viral handling...When working with lentivirus, many organizations require or recommend disposable gowns over your lab coats... provided. Eye protection or face shields are required whenever there’s a splash or spray risk in BSL...BSL-1. In BSL-2 labs, eye protection is required any time you have to work with BSL-2 materials, such as...working in a biosafety cabinet, even if it is not required by their organization. It’s always okay to add...

... a lot of the “soft skills” required for teamwork may not be a requirement. Your manager: Unlike academia...Many companies will hire a postdoc to perform a specific project, while others will hire a group of postdocs...working as a team to develop therapies that could directly impact patients. And, to be honest, I’ve never...be motivated to get your project in a workable direction quickly. Of course, you might have less time to...industry experience” that I’ve seen listed as a requirement on some industry job postings. Also, if you work...academic setting—especially if job applications require teaching experience or high numbers of publications...publications, which may take a longer time to acquire. Learning about drug discovery: Unless your academic lab...

...producing firefly luciferase. Firefly luciferase can readily be measured to quantify (indirectly) transcription...minigenome systems, such as the one developed for Zaire ebola virus (EBOV) are extremely useful, allowing...contains the 3’ and 5’ ends of the genome which are required for replication and transcription (Figure 1). ...replaced by a single (non-viral) reporter gene (e.g. firefly luciferase or eGFP) flanked by the EBOV-specific...minigenome contains all cis-acting signals necessary to direct minigenome replication and reporter gene transcription...reviewed in 2, 3). Other important components of the Zaire ebola virus minigenome system Similar to other RNA...transcribes viral genes. Four viral proteins are required for viral genome replication and transcription...

...order and direction (which is not easy to see in colony PCR electrophoresis), is free of undesired mutations...achieve before starting your experiments. Don’t dive directly into the pipetting! MoClo nomenclature and structure...for new users, and your cloning success depends directly on the choices you make during the early stages...Know your process The MoClo system allows for directional assembly of DNA sequences, called modules or ...Assembly is dictated by four-base overhangs that are paired with different part types (e.g. 5’-GGAG and 3’-...adequate overhangs is crucial for the position and directionality of the sequence you are trying to clone. ... way your synthesized DNA fragment can be used directly in your assemblies. Preparing reagents Keep it...

...start by looking at the requirements in the job description. For each requirement, ask yourself if you have...the job requirements, strengthened an application. Even so, there were examples of great hires who hadn...Similarly, entry-level jobs are less likely to require cover letters, while higher-level jobs are more...submitted for their entry-level jobs and does not require or request them for these positions. (They are ...how your skills and experience match the job requirements, by giving specific examples of your work and.... Let’s imagine you’re applying to a job that requires ddPCR skills. Your resume lists ddPCR, so the ... explain how your experience matches the job requirements, and address any unusual circumstances or proactively...

...spreading through the neuroscience community like wildfire and for good reason. For the first time in the...spent two months in the lab of Antonello Bonci, Director of the National Institutes of Drug Abuse Intramural...m not an electrophysiologist so I cannot speak directly on this topic but, in numerous studies, optogenetics...optogenetics is used in slice physiology in order to directly show that, for example, the firing of a specific...appropriate test.  Of course, proper controls are required and include, for example, confirmation of opsin...optogenetics experiment and what are the tools required for each one? Behavioral Optogenetics can be broken... the optogenetic constructs Expression of your desired optogenetic construct in the brain region and cell...

... Set up a table of contents in the top directory: A directory is a file system in the computer and is...including background on other analyses that have inspired you to perform this analysis. Having a clearly...name for a folder. For instance, your Desktop directory contains the folders and files that you see on...on your desktop. The location of your master directory or folder for all of your analyses is up to you... (see above). Give every experiment its own directory: In addition to output files, this is also where...README.txt document is invaluable. In the top directory of every experiment, immediately write a text ...text document with a brief description of the directory contents, at a minimum. This may or may not be where...

...appropriately edited for your desired mutation - insertion, deletion, or site-directed knock-in. We’ll also discuss...know a few things about your target cells and the desired edits. First, you will need to know what the ploidy...knockout mutations When a knockout cell line is desired, any mutation that introduces a frameshift in the...gRNA and Cas9 combo and are hoping for homology-directed repair events, or maybe you’re performing a single...the best screening method depends on what your desired edit is. Large knock-ins Are you introducing a...may be the way to go. Design a PCR to flank your desired edit site and run the PCR product on a gel to look...mutation in a neighboring nucleotide along with your desired edit. This silent mutation should be designed solely...

...demands of using these wireless devices are significantly higher than standard “wired” optogenetics but may...difficult portion of optogenetics (though it does require a significant amount of equipment, most of which...written outlining the different steps and materials required for each [1, 2]. This protocol [3] provides, in... in exquisite detail, the various steps required to produce your fiber optic implant. Companies such as...the ferrule implant must be maintained during the entire protocol, otherwise optical stimulation will be...primary one is that some behavioral experiments require stimulation only under certain contexts/locations...without a loss of laser power. More recently, wirelessly-controlled optogenetic implants have been reported...

...scientists should acquire during their PhD and postdoctoral training. Addgene executive director Joanne Kamens...were many changes to the  Board of Directors and the Executive Director changed twice. I had to learn how... to report to the Board of Directors and liaise with the Executive Director. As members changed, I had...official assessment and discussion with the Board of Directors, the Board officially voted me in as Chair of ...Different leadership styles Being an effective leader requires you to understand how to work with many different...format depending on the particular style and requirements of the new Board Members. In some instances .... Some members would need much more guidance, direction, and a clearly laid out plan of action, others...

... techniques have in common? They all typically require secondary antibodies, frequently of the mouse or...certainly aren’t “broken,” their production does require continued animal sacrifice. Could there be an alternative...Antibodies One of the main reasons antibodies are great indirect immunodetection tools is their strong signal strength...expressed in bacteria, which means their production requires animal sacrifice and precludes genetic engineering...one primary antibody. This process can be avoided direct antibody fluorophore conjugates are used, but these...dyes Commercially available Not recombinant; Require animal sacrifice Poor tissue penetration Less ...Less resolution due to larger label displacement Require separate incubation of 1° and 2° Must use different...

...Limited genomic integration Multiple transfections required Need to sequence to verify there’s no genomic ...Transgene-free and vector-free Multiple transfections required Protein Delivery DNA-Free Low High Transgene-free...they have several safety concerns. First, they require the use of potentially harmful viral particles ...integrating viral systems currently lack the safety required for translational use. Viral Vectors Retroviral...transposon insertion site, so sequence-verification is required. Non-integrating iPSC delivery methods Viral...promoters for transcriptional regulation, so it requires a different approach for regulating transgene ...Generating iPSCs with plasmid-based expression requires serial transfection of 1 or 2 plasmids that express...

...and not require interaction with the data authors. In some types of research, raw data requires huge amounts...Since early March, the open access publisher has required authors to include a note as to where readers ... formal statement that is already practiced or required by many other journals and several funding agencies...statistical methods, omission of some data, etc.  The firestorm that followed implementation of the policy comprised...be accessed. Adding to this burden, the policy requires that data should be available for anonymous request...evaluate the interpretation of the experiment would require access to each individual video. To PLOS’s credit...not finalized its definition of necessary data requirements and likely will not do so (calling it a “sisyphean...

...sites so that scientists can easily clone in their desired DNA sequences. Gateway technology relies on the...coli strains, most colonies should contain the desired, recombined construct. Read our recent Plasmids...where we might use Gateway cloning to generate our desired constructs: lentiviral expression of the human ...There are a few different ways to generate our desired entry clone - human KRAS flanked by attL sites....donor vector.   Method B: TOPO-cloning of the desired insert into an attL-entry-TOPO vector. TOPO cloning...depend on a number of factors, like your organism, desired expression level, and experimental purpose. For...and destination vector creates two products: the desires expression clone and a byproduct containing the...

...limited by the low editing efficiency of homology-directed repair (HDR), the pathway for resolving DNA double-strand...backbone, sidestepping the problem of HDR vs. NHEJ entirely. The first two classes of base editors were cytosine...changes. Solid red arrows indicate a conversion directly catalyzed by the base editor. Dotted red arrows...does base editing work? Base transition editing requires three elements. They are, broadly: A Cas protein...fourth-generation base editors, BE4, reduce the undesired C→G or C→A conversions that can happen with BE3s...editing efficiency (Koblan et al., 2018). Later directed evolution efforts yielded an editor with more ...et al., 2017). Creating an adenine base editor required an additional step because there are no known ...

...travel be required or will you stay in one location? How long is the commute? Will the job require public...Science careers divided by the most pertinent skills required Research skills The ability to think critically...and setbacks. There are many roles that actively require these skills, including: Science Reporter Scientific...accuracy and attention to detail are valuable skills acquired from a science degree that can be applied to many... Experience in the field or in a position that requires travel can show that you are self organised, reliable... Zoological Worker Conservationist Documentary Director Parasitology Botanist Nature Photographer/Artist...employers. Increasingly, computer skills are highly desired, as roles are becoming more reliant on technology...

...into proteins are steps in that direction (Chatterjee et al., 2013; Hilaire et al., 2017). GFP can only be...myristoylation requires an N-terminal consensus sequence (MG-) and prenylation requires a C-terminal consensus... 2002). GFP needs oxygen Chromophore formation requires a fully folded beta-barrel structure, followed...chromophore (Tsien, 1998). Importantly, this reaction requires molecular oxygen and as a consequence GFP remains...proteins.Therefore, GFP is not suitable for applications that require anaerobic conditions. In experiments requiring ...are generally added to the bathing solution and require uptake by the cells. Which of these strategies ... et al. (2016). To date, small probes that are entirely genetically encoded do not exist, but the development...

...government cannot declare that gravity change direction and make things fly up when we drop them. Scientific... their side of the bargain and do the research required to inform the public (8–13). Facing the possibility...consequences While frustration and anxiety have inspired people to rally for science, others are concerned... to take place around the world. There is no requirement that marchers or supporters be scientists – on...science is done by people like them. This, when paired with long-term science outreach, may have the most...treatments promised by modern research, more funding is required. This is by no means an exhaustive list and every...science rallies scientists to political action, and inspires politicians and citizens to include science in...

...targeted Cas9 cleavage, and repair through homology directed repair (HDR) and non-homologous end joining (NHEJ...created SRIRACCHA in 2017 to enrich and identify desired CRISPR mutants (Wen et al., 2018). SRIRACCHA uses...constructs for the reporter to work and to make the desired genome edit:  A donor plasmid that contains a ...OH-T and doxycycline. A gRNA that targets your desired gene (whose sequence is also found in the reporter...the reporter gene that was cleaved by Cas9 and repaired via HDR using the supplied donor plasmid will ...presumably the endogenous target site will be cut and repaired by NHEJ as well. The ratio of RFP to GFP expressing...integrated GFP reporter leaving you with just your desired genomic edit.  Find SRIRACCHA plasmids at Addgene...

...correspondence via e-mail throughout the entire year. In addition to their direct training role as mentors to Spanish...Co-founder/director of the non-profit foundation IMFAHE and its International Mentoring Program Director of Strategic... as an idea was conceived and developed by its Director, Dr. Zafira Castano Corsino, a junior faculty ...’s Hospital (BWH). Based on her experience co-directing Mentor Circle Programs at BWH and providing career...supported, and can more fully identify with and be inspired by the mentor’s successful example. For evidence...mentors also facilitated the development of bidirectional USA-Europe professional collaborations, setting...their pre-program self-assesment. Achievements directly attributable to participation in the program included...

...enzymes, which have a flexible or non-existent PAM requirement, and how these proteins are advantageous in today...sequence is suitable, but there is no PAM next to the desired cut site, then a further-away gRNA where there ... may wonder: how close is close enough for the desired edit to still occur if the cut site is sub-optimal...it depends, but generally the efficiency of the desired edit is reduced by over 50% when the cut site is...there is a need to eliminate or reduce the PAM requirement to allow flexibility in gRNA selection and subsequent...constraints, or at least has looser or different requirements. Luckily, there are some available.   Cas proteins... generating Cas enzymes with alternative PAM requirements (Zhao et. al, 2023). xCas9 has been shown to...

... Park.  In addition to Taq DNA polymerase, PCR requires free nucleotides (dNTPs), template DNA to amplify...are crucial for this process as DNA polymerases require an existing strand of DNA to add nucleotides to...exponentially replicate the target DNA of interest. This entire reaction used to be done manually in water baths..., or for generating mutated sequences for site-directed mutagenesis.  Quantitative polymerase chain reaction...measuring a starting DNA concentration using PCR. qPCR requires the addition of a probe based fluorescent dye ...transcription-qPCR (RT-qPCR). The first step of this process requires RNA to be converted to cDNA using reverse transcription...rare alleles or mutations. ddPCR also does not require a reference or standard curve, which can be time...

...currently requires additional experimental manipulation. In some cases, injection of Cre is required, however...selection marker (Kim et al., 2014). Meanwhile Andy Fire’s lab used oligo-mediated knock-in of a dominant ...development of this approach reported in 2017 uses the direct injection of a CRISPR/Cas9 ribonucleoprotein complex...advantage of being used in any genetic background, but require variable amounts of experimental manipulation and...– to work with inefficient sgRNAs in my initial direct screening efforts, which were similar to the approach...products used to identify a single 2xFLAG knock-in by direct screening. The center images depict the power of... DNA double-strand breaks up to 54 bp from the desired insertion site result in efficient editing. Repair...

... homology directed repair (HDR) to incorporate new information into DNA. This also requires an exogenous...lightly! Indeed, truly achieving the gold standard requires not one but two rounds of single cell cloning ... of target site is far more constrained by the desired location of edit; efficiency decreases dramatically...limited to single nucleotide transitions but still requires a nearby PAM, although these are still early days...to optimize numerous parameters to generate the desired edit. The wrench: Gene activation and inhibition...technologies – a nuclease-dead Cas9 (dCas9) is directed near the promoter of a target gene. Here, the ...the FANTOM database, which relies on CAGE-seq to directly capture the mRNA cap, provides the most accurate...

...-use of existing elements. DNA sequences for a desired component are cloned into Golden Gate entry vectors...expression to degradation. Chemical approaches may require expensive peptides or small molecules and exceed...on or off the expression of shRNA. This system requires less work than using established methods and minimizes...thorough understanding of complex biological systems requires both a catalog of molecular players and contextualized...fragments contain 482bp of overlapping sequence that direct Homologous Recombination (HR) or Single Strand ...strain EcAR7) and minimizing the set of plasmids required to make singly or multiply phosphorylated proteins...orientation, so that multiple DNA components can directionally assemble in a single reaction, while retaining...

... in Gene Therapy, Director of the Grousbeck Gene Therapy Center, Associate Director of the Ocular Genomics...Addgene welcomes three new members to the Board of Directors: Khandan Baradaran, Frank Stegmeier, and Luk Vandenberghe...of Quality at Dimension Therapeutics, which was acquired by Ultragenyx Pharmaceutical in 2017. As one of...In 11th grade she heard a talk about Z DNA that inspired her to major in molecular biology in college. ...funded,” she says. Baradaran met Addgene Executive Director Joanne Kamens several years ago through the Healthcare... an HIV lab and based on that experience, soon directed himself to finding “immunological and virological...commercialization efforts in gene therapy either through direct industry collaborations, grants, or setting up ...