...implanted into a female animal for gestation. Nucleases that induce double-stranded DNA breaks, such as...cells, which are then injected into a blastocyst. Nucleases can also be used to engineer knockout mice. ZFN...non-homologous repair at the site of DNA damage. These nucleases are microinjected into a zygote, which is then...introduce point mutations. While using targeted nucleases to generate an engineered mouse model has advantages...targeting vector alone, some challenges still exist. Nucleases may cause off-target effects on regions of the... random process, zygotes edited using the same nuclease may not result in genetically identical mice. ...about Cre-lox Read our Genome Engineering Guide Learn more in our CRIPSR guide ...

...mEosFP exposure to UV light causes an irreversible cleavage near the chromophore, resulting in emission of...to blue light also inactivates the chromophore, leading to a non-fluorescent state. Dronpa can be reversibly...quickly inactivated and less photons are emitted leading to dimmer fluorescence. In fact, many of the early...are being emitted, making it easier to capture a clear image above background. The majority of PA-FPs come...disrupting cellular function and localization, or leading to protein aggregation. Another important parameter...light used to activate the first PA-FP does not photobleach the second, and vice versa. Finally, as every...contrast, PA-FPs offer a reduced potential for photobleaching and phototoxicity that standard FPs might induce...

...including mitosis, mitochondrial fission/fusion, nuclear import, and neuronal trafficking. Although FPs ...Overexpression of fluorescently tagged proteins can lead to improper protein localization, protein aggregation...power of this system lies in the ability of the endonuclease Cas9 to create a DNA double stranded break (...the tag, selectable marker, and connector to be released (Fig. 1, Step 2-3). Since donor plasmids of the...directed repair after a CRISPR/Cas9 induced break. Learn more about these constructs and the cell lines they...Cas systems used for endogenous protein tagging, please visit our website CRISPR/Cas Plasmids - Protein...PMC4889673. Additional Resources on the Addgene Blog Learn about Fluorescent Protein Timers Check out Photoactivatable...

...DNA could lead to transgene expression in recombinase-negative animals, producing misleading results.  ...the plasmid used to create the viral prep. In a clean viral prep, like AAV-44362 shown below, more than...from these known impurities, as was the case for clean sample AAV-44362. In addition to these expected ...number of hits to the same sequence (gene). In a clean sample, there are typically fewer than 10 hits to...receive the best AAV vectors for your research. Please use the comments section below to let us know if...Topic Page Subscribe to Viral Vectors Blog Posts Learn How We Use NGS for Plasmid Quality Control Resources...

... Developed by David Liu and colleagues, FokI-dCas9 expresses Fok1 nuclease domain fused to catalytically... to this system by engineering cells that lack release factor one (RF-1; Bacterial strain EcAR7) and minimizing...control for your experiments. For more information, please see Addgene’s information page for the Rinehart...neuronal signaling. For more information on DREADDs, please see the Roth lab's DREADD users blog. New ... expression plasmid that was used by Chen and colleagues for genome editing inXenopus tropicalis. (Guo...information on using the Platinum Gate TALEN Kit, please see the Yamamoto lab’s protocol for TALEN construction...mGRASP A Nature 2012 article by Jinny Kim and colleagues describes their efforts to map the location and...

...to see the dirty laundry exposed here, but it’s unclear how much they will actually care. The section of...technology. Though these case studies are “old,” they lead Zimmer to one of his most critical observations:...detection had over 1,000 authors. The chapter on deep learning addresses the dilemma of creating a technology...discussed The State of Science with the five Addgene colleagues who read it with me, the consistent response ...non-scientist alike was that the book left them wanting to learn more. There are individual paragraphs that may have...task to set oneself. As a scientist who enjoys learning a little bit about many different things (part...about a long time. For the scientists who like to learn everything about one specific topic, The State of...

... clinical gene therapy applications. Read on to learn more about the current (and future) applications... co-transfection of these plasmids, 293T cells released transgene-containing lentiviral particles into...and they showed that expression persisted for at least thirty days. Although adeno-associated viral vectors...activation from the 5’ LTR) and Rev (facilitates nuclear export of transcripts). A different viral envelope...safer, this system may be more cumbersome to use and lead to lower viral titers due to higher plasmid number...Addgene Blog Your Lentiviral Plasmid FAQs Answered Learn How Lentiviral CRISPR Libraries Enable Genome-Scale... on Addgene.org Check out Our Lentivirus Guide Learn about Virus Biosafety Find Lentiviral Vectors for...

...several washes, this tag can be cleaved by the bdNEDP1 protease, thus releasing purified nanobodies from the...common protein purification methods. Read on to learn more about nanobodies and how their structure and...primary antibodies raised in different species, or at least from different IgG subclasses, so that secondary... and the target antigen to about 2 nm, which can lead to higher resolution for super-resolution microscopy...rabbit nanobodies. If you’re interested, you can learn more about how nanobodies are identified in this...University Use Nanobodies to Control Protein Activity Learn more about protein tags Read about E. coli strains...

...journals. In addition, it is my pleasure to interact with my colleagues in meetings of scientific societies..."Biology is Method", i.e. new technologies often lead to big discoveries in biology. Therefore, my lab...translocation trap (ITT) system to analyze signal-induced nuclear translocation of proteins in 2004. In addition,...much enjoyed interactions with many excellent colleagues at NYU and visiting scientists, such as seminar..., I've been very lucky to work with excellent colleagues in my lab. On the other hand, I feel that researchers... conventional research. So, I hope my younger colleagues may be able to develop new technologies, hopefully...

...the progress of the project. After that rather unpleasant experience I thought: I could do this job so ...and disadvantages of this overall rewarding role. Learn about Other Addgenies and Their Careers in Our Career...Technical Correspondent or similar. While there isn’t a clear distinction between these job titles, they may be...particularly tricky cases, team discussions can often lead to otherwise unobtainable solutions. To some extent...support can add a variety of skills to your CV and lead to other job opportunities (though it is not uncommon...Occasionally it can also be necessary to take the lead and be assertive. You develop an understanding of... Now? Academic v. Non-Academic Career Decisions Learn about Science Career Options Explore Tips for Technical...

...define your scope from the onset. Perhaps you, a colleague, or your adviser was invited to write on a particular...to get a sense of the themes in the field and to learn what people cared about. I also used reviews to ...beginning I felt like this was cheating. Eventually I learned that it’s practical because there simply isn’t ...enough time to read every single paper. I also learned to appreciate the discussion sections of the primary... down and get anything done, so unless I had at least a two hour block, I wouldn’t work on the review....free two hour block of time was. Eventually, I learned that I was most productive in short bursts (even... could find the original paper if you wanted to learn more. Another example: we made a schematic of all...

... science trainees still have to lead on their own development. Please see the AMA itself for all sorts... a great way to establish yourself as a thought leader and have real conversations with other scientists...forums and try to keep discussions on-topic and, at least sometimes, civil. r/Science in particular hosts ...answered many questions in the AMA but, as you’ll learn below, even with many resources at her disposal,...potentially hostile posters (this advice included leaving out the term “implicit bias” which is apparently...academic one for the majority of scientists and I am pleased to see graduate and postdoc programs beginning ...

...Previous Post on CRISPR Mouse Models Learn about Homology Directed Repair Learn about Cas9 Ribonucleoprotein ...previous methods yielding 1-10% knock-in. Read on to learn how you can make CRISPR mouse model generation easi-er...re fortunate that there’s a guide that directs cleavage right before the stop codon!   Design your repair...Miura et al. (2018) recommend targeting sites at least 100 bp away from the target exon on either side....in a two-guide floxed experiment, if one guide cleaves less efficiently than the other, a partial insertion...situ germline genome engineering using CRISPR nucleases. Genome Biol. 19 (2018) (1):25. DOI: 10.1186/s13059...

...High School visited our labs in Cambridge, MA to learn about the wide variety of science careers that Addgenies...that it’s never too early to start networking! Learning about careers at Addgene Afterwards, the students...: “Kory was awesome!”. The students also got to learn about intellectual property from our Tech Transfer...career paths, Michelle Healy said “You are always learning and that means it is possible to switch your field...Additional Resources Visit the MassBioEd Website Learn more about Dennis-Yarmouth Regional High School ... Blog Posts Get Career Advice from Our Bloggers Learn How to Improve Your Networking Skills  ...

...You wouldn’t leave your oven on all day to save time making dinner at night, so why leave your lab equipment...Addgene blog Learn about recycling initiatives for the lab Subscribe to the Addgene blog Learn about other...difference? Individual changes to our behavior can lead to cultural shifts that can have bigger impacts....even one less run a week makes a big difference. Clean out freezers so you can make room and don’t have...

...nonimmigrants). The foreign worker must possess at least a bachelor's degree or its equivalent and state ...books, journals online and/or in print. Having a lead role or critical role at a prestigious institution...successes in the performing arts- not relevant at all. Please do not include the fact that you are a classically...company you must show that your company employs at least 3 full-time people engaged in researcher activities...from the point of view of experts in your field. Please remember, this is not a job reference letter. This...awards, presentations, in .pdf format.  Last but not least, NETWORK! Networking is extremely important for ...business development, finance, and operations. To learn more, visit www.propelcareers.com. Casseus Law: ...

...perhaps there is a clear winner, is there something that we can invest our time into learning and extract the...timers: You need to learn the language and what the parameters represent. The best learn-by-doing tool is...for improvement and a year later it became quite clear that others thought the same. Since January 2013...microhomology aware”. Trends: Fewer tools are being released with advanced functionality, it seems these kinds...blogger Cameron MacPherson! Cameron R. MacPherson is Lead Data Scientist for the Milieu Intérieur project,..."DNA targeting specificity of RNA-guided Cas9 nucleases." Nature biotechnology 31.9 (2013): 827-832. PubMed...

...fluorescence. In Brainbow 2.1, GFP is tagged with a nuclear localization sequence, allowing it to localize ...mouse generates cells that randomly express either nuclear GFP, membrane CFP, cytoplasmic YFP, or cytoplasmic...Cre undergoes exactly one recombination event, leading to each copy of the transgene expressing either... tags instead of XFP to optimize it for tissue clearing, a method that makes tissues, such as the brain...visible. Chemical tags are more resistant to tissue clearing methods and can be labeled by synthetic fluorophores...blog Read an introduction to live cell imaging Learn more about the origin of the Brainbow system Visit...Browse Addgene’s fluorescent protein collection Learn about fluorescent tools for in vivo imaging ...

...this gap by both highlighting that researchers do learn MANY transferable skills while working in the lab...have the best time management skills, but I’ve learned a lot since my days as an undergrad working with... variety of projects. I was therefore forced to learn a variety of ways to manage my time. As a busy researcher...distractions: Probably the easiest way to do this is to leave your phone in your bag on “Do Not Disturb” during... recommend the book, “Bored and Brilliant” for learning more fantastic reasons to remove yourself from...you have any other time management suggestions, please be sure to note them in the comments! Additional...Get Advice on Building a Grad Student Community Learn about Careers in Science Communication Resources...

...Scientists enjoy learning new things. Building new relationships is all about learning new things from ...scientists-in-training rarely get the opportunity to learn about the myriad of diverse, nonacademic careers...worth!) with academic scientists who can’t help us learn about the nonacademic sphere where most of us will...really, always). Informational Interviews are for learning and for expanding your web of connections. A mistake... are always setting up your next opportunity to learn. Additional Reading More "Career Advice for Scientists...