...optimized, it’s important to ascertain how long the rescue phenotype will last, and more importantly, if it does...genetic disease in a postnatal mouse model, an important proof of concept for future preclinical and clinical... time, which would prevent the accumulation of unwanted mutations over time. DMD is a good choice for ...does not cause obvious germline modification, an important finding given the controversy associated with ...great step forward for CRISPR gene therapy, it’s important to realize that DMD is a simpler case than other...and large animal models with mutations in human-relevant exons 45-55, rather than in the traditional exon...Potential off-target effects in muscle, as well as unwanted germline editing, must be rigorously examined ...

...impractical to catalog every variant of a feature. Instead, we identified common variants, and then crafted a ...principles. For every task, we envision what the user wants to do and make the path to accomplishing their goals...with every possible option, we place the most important controls front and center, and make specialized...sequences. These plasmids contain features such as antibiotic resistance markers and replication origins, but...threshold. This issue is addressed by adding more variants to the database. Another limitation is that by...augment the database with new fluorescent protein variants. The future of DNA feature identification What...to newly sequenced strains through our mismatch tolerant detection algorithm and thereby speed up the annotation...

...16528756. 2. Oh, Steve KW, et al. "Substantial overproduction of antibodies by applying osmotic pressure and...Rather than being released into the cell culture supernatant, the viral particles associate with the cell ...induce hypertonicity display enhanced monoclonal antibody production (Oyaas, 1989; Ozturk and Palsson, 1991...scientists rely on our AAV preparations for, we wanted to be certain that this supplementation strategy...test this approach with different capsids. Most importantly, because our AAV yields are improved we will ...18613082. 3. Oyaas, K., et al. "Hybridoma growth and antibody production under conditions of hyperosmotic stress...osmolarity on hybridoma growth, metabolism, and antibody production." Biotechnology and bioengineering ...

...going to tell you to pick up the pace Constant worry about grants and renewals What is different about...scientists start out in academia, it is especially important for graduate students and postdocs to get a perspective...interesting topics Having strong non-science skills is important for success Must always justify use of resources...outsource a task someone else who can do better, no grant writing Job changes can happen regularly (every ...learn about the diverse options and how to take advantage of them early is one way to smooth your path to...scientists following diverse career paths is an important first step. Developing mentoring relationships...enjoy the most. Having a better idea of where you want to go will help you focus on the transferable skills...

...background. In other cases, YFG or one of its mutant variants will need to be overexpressed or knocked down...disrupts the function of YFG. You could compare this mutant fly to one lacking the mutation in the same genetic...from having to create a new fly line every time you want to express YFG in a new tissue type (more details...For example, if you discovered five new genes and wanted to express them within a specific neuronal compartment...eg. LexA/LexAop and Cre/Lox systems) in case you want to express one gene in one cell type and another...process of generating your own fly lines. In the meantime, be sure to browse the publication references ... a means of altering cell fates and generating dominant phenotypes., Development 118, 401–15 (1993). PubMed...

...reaction. Table 1 compares some general advantages and disadvantages of luminescence versus fluorescence....studies utilized luciferase as a genetic reporter in plant and mammalian cells. Luciferase assays have since...Used In Concert with Fluorecent Proteins in Nano Lanterns Reporter gene assays A reporter gene, such as...bright than fluorescent proteins, but have the advantage of lower background levels and improved signal...expression. The regulatory element (wild-type or mutant) is cloned upstream of the luciferase gene in the... is then introduced into the desired mammalian, plant or bacterial cells. Expression of luciferase as ...and the activity measurement can then be used to quantify the effect of the regulatory element. Depending...

...a PhD student in his first year. “It was very important to me to be with them in the lab when they started...the lab and to take things slowly! It is more important to carefully select first hires who are well-suited... having projects ambitious enough that everyone wants to recruit rather than repel and hoard.” If you'... prepared me for what a PI without a personal assistant has to deal with. My inbox currently stands at.... “Being a PI is more than just experiments and grant writing. You have to enjoy the mentoring role.” ...every day and in the pub with us in the evening, wanting to know our latest results as soon as the gels ... build a strong, collaborative team. And take advantage of the many resources available from your institution...

...does one stand out amongst a pool of successful applicants and get chosen to work in his or her lab of choice...This is followed by some perhaps surprising, yet important, factors to consider when seeking your dream lab...the type of science we do fits in with what they want to do. The experiments must fit with their interests...short-term goals and chat about awesome science. Important factors in the search for a lab Studies show that...postdoc. Although the chosen research field is important, the end goal during this time is to be trained...role models and teach you to be the scientist you want to be. For much more on this topic see our blog ...neighbors during the interview process. What if I want a non-academic career path?  If you are considering...

...should I take to get to work?”; we often make important life decisions without much logic - even choosing... of work and communication before making this important decision. I’ll highlight some of these questions... if it is what is best for you. Some of these important questions will be easy to ask the lab head, and...the mentors you choose are teaching the stuff you want to know and display character traits and skills ...the department? Are they successful at getting grants and funding? Beware of bullying Your mental health...your aspiration? Will you help or at least observe grant writing? Get equipment quotes? Understand administrative... a career at a small, teaching college you might want to consider less expensive, portable areas of research...

...University of Amsterdam. Before you decide which car you want to buy, it is worthwhile to test-drive a couple ...the best advice is to try a couple of promising variants to check how they perform under your experimental...protein properties need to be scrutinized under relevant conditions. The properties of fluorescent proteins...detected signal and therefore high brightness is an important feature of a fluorophore. The key question is ...indicated by the extinction coefficient (EC) and quantum yield (QY) respectively. The theoretical brightness...However, the theoretical brightness ignores several important experimental conditions that are related to the...fluorescent protein in the host cell, which is a key determinant of the practical brightness. Therefore, practical...

...Mentoring Group all participants act as both mentors and mentees. Because the participants are at a similar...make mentoring less mysterious and train the participants in skills useful for future mentoring relationships...step-by-step, how-to guide to get one started. Advantages of the group mentoring format Adding peer advice...at the same time Broadens the network of the participants Can be a lot of fun! Form your own peer mentoring...talk to trainees more about peer mentoring. They want to find mentors to help them develop their skills...Discuss your general areas of interest and what you want to cover during the year. Suggest that each person...perhaps a 3 hour breakfast once a month. All participants must commit to making the meetings a scheduling...

...reasons why doing something without a kit has advantages: less plastic waste, less expense, and less of...such as RNaseZAP. When harvesting tissues, cells, plants, fungi, or bacteria, keep samples cold and work...effectively isolate intact RNA, a fast, strong protein denaturant would be required- something that broke down ...Chirgwin and colleagues showed that a strong protein denaturant, guanidinium thiocyanate, did just this (Chirgwin...Chirgwin et al., 1979). They developed a protocol meant for isolating RNA from rat spleens in which they...material is removed via centrifugation and the supernatant is extracted with chloroform as in the above ... results of a downstream application such as quantitative PCR to assess gene expression. There are several...

...from countless rounds of optimization. Dozens of variants exist with unique cutting features and evolved...engineering endeavors. For more information on Cas9 variants, check out our blog post on choosing a Cas9 enzyme...effective at wiping out large regions of DNA and has anti-viral application potential as a result. Fast facts...enzymes outlined below as well. Cas12a also has significant therapeutic promise due to the fact that it is...target sequence Similar to Cas12, it is also significantly smaller in size than traditional Cas9. Another...when engaged by its target sequence. Why would you want to give it a go? The high specificity of Cas14 makes... detection tool for rare, often hard to detect variants such as single nucleotide polymorphisms. Fast...

...al. were interested in correcting an autosomal dominant mutation in MYBPC3 that leads to hypertrophic ...in cases of disease prevention. However, it is important to consider that already-tested CRISPR alternatives...cardiomyopathy and other genetic disorders, pre-implantation genetic diagnosis (PGD) screens embryos to find... find those that do not carry a given dominant disease-causing gene, or that carry only one copy of a ...clinical applications could be considered. It is important to consider the question of broader applicability...had few predicted off-target effects, but this advantage likely won’t transfer to all genes we’d like to... large step forward in the CRISPR field, it’s important to remember that the study hasn’t achieved any...

...for oxidizing environments to photoconvertible variants, it seems like GFP is always learning new things...frustration many researchers have experienced: wanting to ask and answer experimental questions, but not...needed to do so. Cepko lab researcher Jonathan Tang wanted to express a variety of proteins in single cell...nanobodies, the project really took off. Unlike most antibodies, nanobodies, which occur naturally in camels,...optogenetics in specific cell populations even easier. Advantages and other possibilities It’s clear that Cre-DOG...GFP-labeled lines, and they each have their own advantages. T-DOGGs can be made drug-inducible using the...proteins with a modular structure, constructing split variants should be relatively straightforward, but it is...

... controlled by regulatory networks in the plant. If we want to access that type of material - or do what... therapeutics, if you want to program a cell to identify and target a malignant cell, then you’d need ...talk about off-target interactions. That’s relevant if you want to make sure you don’t target anywhere else... parts are required to get the function that you want. The last few years, we’ve really been making tons... sophisticated. There are also proteins, like antibodies. But we are really just using what biology has...

...set out to make my first CRISPR/Cas9 gene edit. I wanted to inactivate the gene BRAF (a kinase implicated...expression in A549 cells transduced with lentiCas9-blast. Anti-Cas9 Western blotting was performed on whole-cell...at the indicated multiplicity of infection (MOI). Anti-vinculin Western blotting is a loading control. ... additional 3 weeks under blasticidin selection. Anti-Cas9 Western blotting was performed on whole-cell...having active Cas9 (see tip 2). Pro-Tips 1. To quantify Cas9 activity using this assay, you really need...of the gene with multiple gRNAs, but the most important is that you could avoid potential off target artifacts...be easier to visualize and identify). If you need/want to put the digestion site in the middle of the PCR...

...fusion protein that switches to any color or tag you want? It sounds like magic, but it’s real! In this post...photophysical properties of chemical dyes provide many advantages over fluorescent proteins, opening up new applications...HaloTag is actually HaloTag7, and recent specialized variants run up to HaloTag11. HaloTag is now the most popular...for every new-and-improved fluorescent protein you want to try, just swap in a new fluorescent ligand and...decorate synthetic viral particles with BG-linked antibodies to drive cell type-specific delivery of a viral...any cloning at all! As mentioned above, several variants of these tags have been developed over the years...are typically 230–250 aa. Smaller size isn’t a guarantee that a tag won’t disrupt the function of the target...

... produce 3 concurrent base edits to withstand antibiotic selection. As a result, 8 mutations in TadA could...modified ABE7.10 accordingly which resulted in variants named “ABE8.x”: “8” stands for the eighth round...x-d.  This resulted altogether in 40 new ABE8 variants. When comparing on-target DNA editing efficiency...replaced S. pyogenes Cas9 in the ABE complex with PAM-variant Cas9 proteins: the S. pyogenes NG-Cas9 (PAM: NG...PAM: NNGRRT) to create Sa-ABE8.x-m/d. With these variants, ABE8 could target many additional locations in...cells using 8 selected ABE8s and ABE7.10. They quantified genomic editing by measuring the reduction in... guide-independent off-target deamination, an important attribute for therapeutic application. ABE8s demonstrate...

... can return multiple answers. You might want to take advantage of their save and recall functions when... with significant lot-to-lot variation. Additionally, this assembly, which used only antibiotic screening...Langhorst, B. W., Bilotti, K., Pryor, J. M., Cantor, E. J., Canton, B., Knight, T. F., Thomas C. Evans, J.,...you get results you like. NEBridge SplitSet If you want to design an overhang set specific to your DNA sequence...J. M., Potapov, V., Kucera, R. B., Bilotti, K., Cantor, E. J., & Lohman, G. J. S. (2020). Enabling one-pot...