...Why the HEK not? References and Resources References Shaw G., et al., Preferential transformation of ...determined it is most likely adrenal in origin. (Anatomy refresher: the adrenal gland is next to the kidney...were isolated from is still a mystery, but it’s currently assumed that the cells came from an aborted fetus...genetically female. HEKs are generally cultured as an adherent monolayer, though derivatives have been adapted...have some neuronal qualities.) While HEKs probably aren’t a great model of kidney cell biology, there are...

...of many complex cellular behaviors, such as differentiation or metabolism. Our attempts to manipulate these...genes or to introduce heterologous genes, but the trend of research has shifted in recent years toward tinkering...system for multidirectional control The CRISPR interference (CRISPRi) model consists of a catalytically ... multiplex control via scRNA design is readily apparent in metabolic engineering. The authors elegantly.... cerevisiae by using three RNA programs to differentially alter the levels of five genes that encode ...sequentially rerouted the metabolic flux to generate differently pigmented derivatives of violacein. Using dCas9...that will still recognize guide RNA but with a different dCas9 binding module, alternative sets of transcriptional...

...available jobs, fields, and trends “Never Leave an Empty Seat” at conferences and seminars, as this is ... path to professorship. But recently a downward trend in funding and space in academic research labs has...traditional academic setting and to shape and strengthen interests and skills. Although the word “extracurricular...potential EAs and which professional skill sets are strengthened. This short list highlights the ubiquity of ...particular field of study Assess your interest in a different field Outreach activities (e.g. Volunteer...Marketing Communication Business models Conference or club organizer Management Leadership...know where graduate students and postdocs are currently getting hired? Let us know what topic you'd like...

...seeing adoption of the technology in different labs for different applications.” Li’s team noted that ... genetic diseases, and several treatments are currently being tested in clinical trials. It is speeding...cells of patients with cystic fibrosis (8).   The difference with CRISPR/Cas9 in comparison to other genome...for selection and optimization. In addition, the current work in developing anti-CRISPR proteins aid the...possible through the use of nanoparticles. [6].    References 1. Zhang B. 2021. “CRISPR/Cas gene therapy.” ...at genome editing with CRISPR-Cas9 technology.” Current genetics. https://doi.org/10.1007/s00294-019-01040...its Application in Genetic Diseases: A Review.” Current pharmaceutical biotechnology. https://doi.org/10.2174...

...of the program beneath it. You can jump to the different time stamps in the description to watch any sections...precisely. You can find a great discussion of different types of replication in Nosek and Errington 2017...another’s reagents with the confidence that they aren’t simply doing an experiment wrong. However, even... a plate or running a gel have intricacies that aren’t always mentioned in static written protocols. It...scientific findings. This makes sense on many different levels - it’s hard to process large amounts of...to get a publication (for better or worse, the currency for success in academic research). Initial positive...review our experimental designs and we need to be transparent about the reasons we alter these designs if we...

.... Unfortunately, there really aren’t any effective off-target reference controls. Off-targets and your...they do arise. Happy CRISPR’ing! References and Resources   References Rabinowitz R, Offen D. Single-Base...effects should impact your experimental design differently. Yes, it is always better to reduce off-target...mutation, off-targets must be quantified slightly differently. For a comprehensive approach, RNA-seq would ...acknowledged as a potential limitation in a study. Strength in numbers If you assess your off-target frequency...to control for off targets as well as clonal differences. If you are performing CRISPR library screens... as a transfection or transduction control, a different gRNA would have distinct off-target sites, eliminating...

...imaging for advanced monitoring of different factors, in different or the same cellular location, all ...covered with packaging plasmids! References and Resources References  Bhalla N., Jolly P., et al. Introduction...biosensors provide quantifiable readouts for levels of different chemicals, hormones, proteins, etc., that you ...and zinc. These ion sensors can be targeted to different cellular locations or tissues depending on the...acceptor of a FRET pair on actinin will produce different signals based on changes to the structure of actinin...organisms, but AAVs make it possible. In AAV systems, different serotypes can be used to affect or determine viral...

...skills that were quite different from mine. Learning to bridge those differences was a pivotal moment in...technical problems in the lab, management involves a different skill set: mentoring, setting strategic goals,... day-to-day details. Ignoring individual differencesManaging a team isn’t “one size fits all”. Each team...team member has unique motivations, strengths, and learning styles. For example, during my time as a lab...autonomously. Failing to recognize and adapt to these differences could have led to frustration, micromanagement...cohesive, productive environment. For example, in my current role managing a cross-functional team on a biomarker...team meeting to highlight their complementary strengths. By encouraging alignment on goals and clarifying...

...isolated from different organisms and engineered Cas9 variants that were able to target different PAM sites...activity of Sniper-Cas9 to different Cas9 variants using gRNAs of different lengths (Lee et al., 2018)...found in other species can recognize a variety of different PAM sequences. Researchers have also used experimental...specificity can target more sites. Created with BioRender.com. Specificity Specificity is the assessment...relative to eSpCas9 and SpCas9-HF across a number of different sites. Using a combined random and directed mutagenesis...choosing a Cas protein for your experiment. References Hu, Johnny H., et al. "Evolved Cas9 variants ...

...performance under different imaging conditions (e.g. different emission filters) or different set-ups. Many...uptake by cells. To correct for this variation, a reference fluorescent protein that is expressed at an equal...be used. An assay developed in our lab uses a reference protein that is translated from the same open-...between the fluorescent protein of interest and the reference protein (Goedhart et al, 2011) allows for a precise... a set of fluorescent proteins (with the same reference protein) under identical conditions, a ranking...for co-expressing mTurquoise2 with mVenus as a reference protein is available from Addgene. Another way...

...Nano-lantern technology. Nano-lanterns contain a Renilla luciferase variant fused to an FP; when supplied...Solving the problems of low luminescence By itself, Renilla luciferase has a low quantum yield, and when transiently... first Nano-lantern, a fusion of a mutagenized Renilla luciferase (RLuc8) with Venus. This luminescent...resonance energy transfer (BRET). Photons emitted by a Renilla luciferase variant are used to excite the fused...technique’s versatility and adaptability to many different contexts. Improving Nano-lanterns In 2015, Takai...characterizing Nano-lanterns and eNL for many different applications. For long-term imaging, especially... for excitation, you instead have to rely on differences in emission wavelength. Filtering the emission...

...ganglia in movement disorders.   References and Resources References Armbruster BN, Li X, Pausch MH, Herlitze...GFP) and mCherry, two of the most common tags currently used, are approximately 240 amino acids in size...in monkey neurons, we next hypothesized that a different tag fused to the hM4Di might improve the transport...Surprisingly, we found that this small change made a big difference. Replacing the mCherry with the HA tag resulted...Overview of Affinity Tags for Protein Purification. Current Protocols in Protein Science 73: . https://doi....doi.org/10.1002/0471140864.ps0909s73 Additional references on the Addgene blog Browse Fluorescent 101 blog...Find more about the many types of protein tags References on Addgene.org Check out Addgene's Chemogenetics...

...any end goal not available with current techniques like PGD.  Reference Ma, Hong, et al. "Correction of...al. designed plasmids containing Cas9 and two different gRNAs, and electroporated them with an ssODN repair...editing through the NHEJ pathway. Created with BioRender.com. In later characterization studies, the ...Moreover, introducing new DNA seems impossible with current tools, thus quieting some fears about the creation...creation of “designer babies.” The differences in editing between iPS cells and embryos make a case for conducting...of such a treatment on top of the expense of IVF render this argument less convincing at present.   Another...embryos to only 60%? Unfortunately, given the differences between CRISPR repair in embryos and iPS models...

...replicates would have different labels - i.e. they would stochastically arise from different cells throughout...arise during treatment or are preexisting in the parent population prior to the start of treatment. They...They hypothesized that, if they labeled the parent cell population using the library, propagated it, separated...labels - i.e. they would always come from the same parent cells that represent the preexisting resistant ... determine using NGS because the error rate of current NGS approaches allows for a detection limit of ...up-front quickly. This approach is in contrast to the current treatment paradigm where resistant populations ...development of combination therapies.         References 1. Hyo-eun, C. Bhang, et al. "Studying clonal...

... use of several Cre transgenic mice targeting different brain areas enabled the drawing of a detailed ...the genOway press release for more information. References Abdallah, Khaled, et al. "Adeno-associated virus... delivery of Cre recombinase in mouse primary afferents." Scientific reports 8.1 (2018): 7321. PubMed ...for analyzing the neural circuits of behavior." Trends in cognitive sciences 15.12 (2011): 592-600. PubMed...Optogenetic approaches to study the mammalian brain." Current opinion in structural biology 57 (2019): 157-163...and Peter Hegemann. "Rhodopsin-regulated calcium currents in Chlamydomonas." Nature 351.6326 (1991): 489...conditional gene modification technology in the mouse." Current protocols in immunology 105.1 (2014): 10-34. PubMed...

...in with the current literature. Finally, if you give your talk or poster to enough different kinds of audiences... NIH, in the United States) Strong letters of reference that attest to your talent and expertise as a ...data needed to rule out alternative models or to strengthen your new model?  PFs need to be regularly thinking...Leader position applications require 3 letters of reference- the graduate school mentor, the postdoctoral ... and your research well. Who could fill these different roles? A second or third mentor. Therefore, seek...the job market. Additional mentors are not so different from having a thesis committee in graduate school...presentations and will be able to provide letters of reference, informal recommendations, more positive grant...

...My name is Lauren Flynn and I am currently wrapping up my co-op with Addgene as a DNA Sequencing Analyst...concepts that are heard often during undergrad. Lauren Flynn is the Addgenie behind our "Meet an Addgenie...and industry jobs. Since there are a variety of different career paths that don’t all need another degree...can have flexibility and be up to date with the current world. Bioinformatics it is.’” Though she misses...Shannon Rinaldi values loving what she does. Her parents told her that if she found something she loves ...-op. It has been so insightful to learn about different career paths, successes and failures, values, ... Lauren Flynn...

...collaboration. The field already has comprehensive reference pages and curated databases for scientists including...discussion on the best strategies and protocols for different experiments.  In this blog post let's take a ...atlas of every C.elegans  neuron. Scientists are currently doing this using two approaches: single-cell RNA-sequencing... peer-reviewed and assigned a DOI that can be referenced. In addition the data published in microPublications...embryonic genes are individually depleted via RNA interference (RNAi). Scientists monitor the effects of RNAi...RNAi using four-dimensional contrast interference (DIC) microscopy and computer imaging processing. The... back frequently for what’s new and popular! References Hammarlund, Marc, et al. "The CeNGEN Project:...

...antibody for your application!  References and resources References Lu, Q. L., & Partridge, T. A. (1998... while polyclonal antibodies contain a mix of different isotypes and subclasses. They each have their ...of clones and thus the potential to bind many different sites on the target protein they tend to have ...polyclonal primary antibody contains a mix of different isotypes and subclasses, use a broadly reactive...secondary antibody binding and therefore the signal strength (Fig. 1). Monoclonal primaries If you do decide... be worth considering an antibody raised in a different species.      Table 1: Isotype considerations...targets, look for primary antibodies raised in different, and distantly related, species, and secondary...

...examine three things: Aligning the NGS result to a reference sequence to confirm backbone elements.   Confirming...deposited plasmids is to align the NGS result to a reference sequence like a known backbone sequence. We’re...through BLAST or by direct alignment to an NCBI reference sequence or a sequence provided by the depositor...synthesized regions that have no publicly available reference sequence or plasmids containing genes with many...the depositing laboratory confirms that these differences are expected and do not affect plasmid function...challenging to QC by NGS as they are often quite different from one library to another. They also differ ... Repository sometime soon. Stay posted!     References Guerin K, Rego M, Bourges D, Ersing I, Haery ...

...library protocols you can use as a reference. Table 1: Examples of Different Pooled Libraries at Addgene.  ... plasmids in your library (Are the inserts of different sizes?), and the desired coverage for the library...library amplification protocol. Created with BioRender.com.   The most important factor to consider...For this reason, if your library has inserts of different sizes (such as a transcription factor overexpression...your growth time (e.g., 16-18 hours). Finding reference protocols There are many decisions to make when...pDNA Library Amplification protocol are useful references. Skim through our Pooled Library page to find...“one-size-fits-all” protocol! Each library is different, and you should always experimentally test the...

... Snapgene and Geneious software. References and resources References Chadeuf G, Ciron C, Moullier P, Salvetti...sequencing reads (~150 bp each) are aligned to the reference sequence of the plasmid used to create the viral...more than 90% of the reads should align to this reference sequence. Second, we perform a megaBLAST search...search on all the reads that did not map to the reference sequence. The resulting list of hits is then carefully...reads into longer DNA contigs without using a reference sequence. The five contigs with the most total...Harten DeMaio K, Sanders E, Tasissa M, Kostman M, Tillgren M, Makana Hanley L, Mueller I, Mitsopoulos A, ... Karen Guerin...

... are composed of many different gRNA-containing plasmids, each with a different genomic target. The goal...1.  What species are your cells derived from? Currently, Addgene carries CRISPR libraries that target ...genome, or a specific class of genes? Addgene currently carries several genome-wide CRISPR libraries and...cell or set of cells containing a mutation in a different gene. Libraries may be available in a 1-plasmid...multiple replicates and several cell types can strengthen your results. Enrichment or depletion of multiple... out our CRISPR Plasmids and Resources Page. References McDade JR, Waxmonsky NC, Swanson LE, Fan M (2016... for Using Pooled Lentiviral CRISPR Libraries. Current Protocols in Molecular Biology 115: . https://doi.org...

...the integrity of different targets and labels. You may want to check out a few different protocols to look...you’ve been looking for too!     References and resources References Bertiaux E, Balestra AC, Bournonville... two points within ~250 nm of each other. For reference, individual presynaptic terminals, which are jam...optically magnify quite so much. Created with biorender.com Steps 2-4 are not found in standard IHC/ICC...traditional full IgG secondaries, can improve signal strength (Sherry and Stiles, 2022). Fourth, signal continuity...2020). Plus, it’s been demonstrated to work in different model systems, paired with SRM techniques, and...

...social life and rest. I need it just as much in my current role as Customer Support and Operations Manager...and projects. It is crucial to identify these strengths within yourself, and to demonstrate them to the...collaboration and fail to identify it as a personal strength until it is identified by another person. Similar...situations where you can learn more about possible strengths and weaknesses, and view everything as a learning...naturally to you. How is this new opportunity much different? Might you already have leadership skills that...solution to the person over email. Examine your current and past job descriptions. Scan through all the...piece of paper and list all the skills that your current or past job requires day-to-day, and you’ll get...

...created with the same backbone but that contain different inserts. When you design primers to anneal outside...primer pair to screen for the presence of many different inserts. The downside: this type of primer does... The type of primers you use depends on your preferences. Either way, make sure to test your colony PCR...a standard PCR thermocycling program. One key difference is the plasmid DNA must be released from the ...and without an insert. These controls are quick references you can use when you run your PCR products out... an appropriate molecular weight standard for reference and to add a loading dye with glycerol to your...products have any mutations.  There are lots of different cloning strategies, but regardless of which is...

..., many plasmids were available, but they often weren’t easily accessible, due to a wide range of logistical...world. This idea, that available and accessible weren’t always the same thing, shaped both Addgene’s founding...distributor for that country. In China, direct shipments weren’t able to clear import control. Partnering with a...form of problem-solving. When universal solutions aren’t enough to ensure accessibility, partnering with...particular topic into beautifully designed and easy-to-reference eBooks. Figure 3: Global readership of the Addgene...which received over two million views from 119 different countries (Figure 4). Four of the five most popular...researchers who use them. The best part of any conference is scientists, from techs to grad students to...

...be translated to other lines. Differences in the physiology of different cell types, however, can make...not only between serum from different manufacturers but also between different lots of serum from the same...concentrated dilutions you will be able to overcome differences in receptor expression. It's all in the mix Even...typically employ the positively charged polycation, polybrene. Interestingly, one study found that alternative...Virus? Check out Addgene's New Viral Service! References 1. Hay, R.J., Macy, M.L. & Chen, T.R. “Mycoplasma...tropism of lentiviral vectors through pseudotyping.” Current Gene Therapy 5.4 (2005). 387-98. Pubmed PMID: 16101513...

...shared ownership. Go check it out! References and Resources References Radtke, A. J., Chu, C. J., Yaniv,...mean highly multiplexed: you can image over 65 different parameters using this method (see Movie S8 from...for spatial biology enthusiasts. This dataset currently includes 1049 community-validated reagents from...fluorescent probes, representing imaging from 47 different tissues. Researchers upload and share both their...resources .csv file for easy filtering and sorting. Reference images can be viewed by clicking on the links ...data, more discussion, and more functionality. Transparency, both in what works and in what doesn’t, and...

...construct containing fluorescent proteins (XFP) of different colors to label an organ or entire organism. Because...localization sequence, allowing it to localize to a different area of the cell than CFP and YFP. The most recent...could recombine to activate the expression of a different fluorescent protein. Therefore, each cell could...Confetti mouse was first used to describe the self-renewal of cells in the intestinal crypt, and since then...proliferating cells. Tetbow optimizes Brainbow for current technologies Tetbow (Sakaguchi et al., 2018) overcame...method that makes tissues, such as the brain, transparent so that the 3D architecture of a fluorescent ...can be combined with new imaging techniques.  References Cai, Dawen, et al. "Improved tools for the Brainbow...