...disparities. But as the science moves forward at a blistering pace, it is becoming ever more urgent for scientists...ways of collaborating, and to organize events that bring different voices together to talk about genetics...running these events will be important both in preparing you for future work in these areas, and in helping...

...approach that problem in a novel way. Finally, engineering new applications to solve problems will provide...’s a good idea to move around to different labs during your career; you learn new techniques and interact...get feedback from the funding body through their scoring system or meeting with their grant submission panel...

...characterized in an unrelated worm (C. elegans) [2]. Delivering dsRNA matching a specific mRNA sequence triggers...and silences its expression. More specifically, during the process of RNAi, an enzyme dubbed Dicer cuts...smaller dsRNA fragments are referred to as small interfering RNAs (siRNA) and they bind to a class of Argonaute...

...here!   Zhang, Z., et al. (2023). Efficient engineering of human and mouse primary cells using peptide-assisted...effective at breaching the blood-brain barrier and delivering a transgene, with neuron bias observed in young...the Gradinaru lab, AAV1-X1, was developed by transferring the 7-mer peptide region from variable region...

...simplicity of gene activation: rather than requiring protein engineering for each loci, CRISPR/Cas systems only...regulation. This can be used to activate transcription during either initiation or elongation, depending on which...

...faster in the following test and skew results measuring locomotion. Mice may need to be given some time...optional handling animals Experiment Behavioral scoring systems (experimenter differences in behavior observation...of our viral vectors blog posts Find tips for preparing AAVs View our Beginner's Guide to Viral Vectors...

...the moderator should be taking good notes and discovering interesting stories, riffs or opinions that each... a fun discussion. People are much better at remembering these things on a relaxed phone call than when...The idea is to ask questions in a directed way to bring out these interesting stories and remind the panelists...

...more gRNAs must be used along the same chromosome. During DNA repair, there is a chance that the two breakpoints...Harnessing type I CRISPR–Cas systems for genome engineering in human cells. Nature Biotechnology, 37(12),...CRISPR-Cas Systems INTEGRATE: Bacterial Genome Engineering Using CRISPR-Transposons Additional resources...

...powerful tool, it was promising enough to keep exploring and to try developing a variant with an enhanced...variants of MagLOV will be possible with other engineering approaches and that magnetosensitive tools can...often posting updates on social media) and freely sharing code, raw data, and more. The team shares results...

... library is maintained and reduce recombination during the amplification process. In liquid culture, all...recombination. If you do see recombination, we recommend ensuring the cells used are meant for repetitive sequences...Addgene blog Genome-Wide Screening Using CRISPR Preparing Pooled Libraries NGS Quality Control for Pooled...

...mass bleaching, the loss of their symbiotic algae, during heat stress. For other organisms, like the sea ... Wistrand-Yuen, P., … Forster, A. C. (2018). Engineering a palette of eukaryotic chromoproteins for bacterial...bacterial synthetic biology. Journal of Biological Engineering, 12(1). https://doi.org/10.1186/s13036-018-0100...

...an industry postdoc is something that you’re considering. Like anything, there are pros and cons to postdocs...number of steps, decisions and people involved is staggering. The process is complicated. If you’re interested...all about teamwork. A single individual could not bring a drug to the market, so people in industry have...

...media with supplements such as magnesium salts, buffering agents, and/or supplying additional carbon sources... yield. Start with a single, fresh colony Sub-culturing directly from a frozen glycerol stock or agar ...harvesting and miniprepping at various time points. Measuring the optical density of the culture can indicate...

... unable to participate in genetic recombination during meiosis. Also, flies are not viable if they are...know is that males do not recombine chromosomes during meiosis. This can sometimes be a handy feature ...Like Addgene, the Drosophila community is highly sharing-oriented, and there are many resources to help ...

...separate proteins should be useful for monitoring cellular events occurring at varied time scales.  Table 1:...the FP timers, add an entire new dimension to monitoring localization; using FP timers, researchers can...

...blastn program uses a smaller word size and lower scoring penalties for mismatches and gaps in the alignment...blastp algorithm parameters. We use the default scoring matrix BLOSUM62, but you may want to check the ... multiple protein sequences, particularly for comparing different isoforms. In addition to our favorites...

...inhibition of the innate immune system; and reconfiguring of the nuclear compartment, gene expression,...’ functions are rescued by helper plasmids used during production and/or by genes present in the HEK293T...license.   The promise of adenoviral vectors This brings us to the final topic, which is that natural AdVs...

... It may be helpful to incorporate regular feeds during this incubation time to maintain cell viability...consider increasing the number of washes performed during your harvest. Validation Recombinant antibodies...temperature shifts. Of course, we are also cautious during the aliquoting and storage process to prevent dropped...

...for distribution. If your experiment involves delivering a calcium biosensor using AAV, you might decide...viral preps instead of plasmids? The available filtering options now change dynamically based on the type...target organism. Clicking through to RNA Editing brings us to a page with a basic overview and figure, ...

... the genome, they could promote oncogenesis by altering local gene expression. Self-inactivating (SIN)... PubMed Central PMCID: PMC4486245. 5. Patient monitoring and follow-up in lentiviral clinical trials. ... Mary Gearing...

...up colony PCR reactions is nearly identical to preparing a standard PCR reaction: combine template, primers...other colony PCR tips are highlighted below. Preparing template: Pick a single colony with a sterile ...to the PCR reaction. The bacteria will be lysed during the initial heating step of the PCR reaction. A...

...requirements. Cpf1’s preferred PAM is 5’-TTN, differing from that of Cas9 (3’-NGG) in both genomic location...repair templates should remove the PAM sequence. Comparing Cpf1 and Cas9 on-target and off-target efficiency... Mary Gearing...

... training, mentoring and coaching services, including Smartegrants, to help those entering the Grants ...Twitter Chat, and then our grant training and mentoring. How do I become a grant professional? Employment...

... the promiscuous cleavage and degradation of neighboring ssRNA and ssDNA by Cas13 and Cas12a, respectively....org/10.1038/s41587-020-0513-4 Chen JS, Ma E, Harrington LB, Da Costa M, Tian X, Palefsky JM, Doudna JA...Essletzbichler P, Dy AJ, Joung J, Verdine V, Donghia N, Daringer NM, Freije CA, Myhrvold C, Bhattacharyya RP, Livny...

... resistance can be the result of one bacterium sharing genes with another, an accidental transmission ...means to defend themselves from these naturally occurring antibiotics. When we use antibiotics to treat ...prescribed to them This may seem counter-intuitive considering the other three scenarios, but a short treatment...

...majority of cell line problems come from people sharing cells: the black market of cell biology. Engaging...cell culture, microbiology, or virology without acquiring materials from this bank. I am often asked, “What...cells to be used and STR profiling to be conducted during projects to ensure the results are reliable. With...

...proteins (FP) have also been important in mapping, monitoring, and manipulating neuronal circuits. Epitope ...tags, low FP expression may be insufficient for monitoring a weakly expressed POI. Spaghetti Monster - A...fluorescent protein structure and color variants." Cold Spring Harbor Protocols 2009.12 (2009): pdb-top63. PubMed...

...subpopulations of cells, you’ll soon find yourself encountering flow data in the literature. Data reported from...of flow cytometry is to use increasingly more stringent markers to identify your specific cell population...are used to separate single cells from doublets, ignoring anything not within the gate of the first plot...

...first non-Cas9 CRISPR protein adapted for genome engineering was Cas12a (formerly Cpf1), a nuclease that generates...comment below. Note: Jennifer Tsang and Mary Gearing contributed to the update of this post. References... genome engineering...

...successful inhibition of hippocampal neurons. Initial monitoring Second, this may sound rudimentary, but before...exciting—and unanticipated—road of discovery. Monitoring behavioral output The appropriate behavioral ...science writing. Derek is also a pianist and an aspiring composer. View his blog drsimonsaysscience.org...

...The first time I heard about FRET during a journal club, my guitarist brain automatically thought about... recently and is the most rigorous method for measuring FRET. FLIM measures the fluorescence decay time...MW. Fluorescent proteins for FRET microscopy: monitoring protein interactions in living cells. Bioessays...

...  Communication (or lack thereof): Information sharing gets more challenging the bigger an organization... can help, and if and when you find yourself transferring this skill to another job, it will be important...conflicts is not inevitable. When you know you are bringing this asset to your job, you can become a valuable...

...Immunofluorescence (IF), is an immunoassay that brings to light the cellular world. The technique allows...poorly in a given context, let your community know! Sharing your experience, positive or negative, will help...Immunofluorescence Staining. In: Methods in Molecular Biology. Springer New York, pp 299–311. https://doi.org/10.1007/...

...sequence in the way that they like, the design and engineering of fusion proteins is relatively straightforward...assessing oligomerization in cells. In addition to measuring stickiness, several researchers have also collected..., Marieke, et al. "A FRET-based biosensor for measuring Gα13 activation in single cells." PloS one 13.3...

...need to test for this in your system if you’re measuring expression over a period of weeks (in vitro) or...to turn on, or turn off, gene expression by administering an inducing agent like doxycycline (Favre et...cells you want to deliver to, and how you’ll be measuring your experimental output. You’ll also want to ...

...Hippocampus labeled with anti-Kv2.1 (magenta), anti-AnkyrinG (green), and Hoechst nuclear stain (cyan). Panels...expect the target to be. You can test this by preparing a sample without the primary antibody, or with...Yong (Ed.), Biobanking (Vol. 1897, pp. 299–311). Springer New York. https://doi.org/10.1007/978-1-4939-8935...

...Matthew L., Sonya J. Wolf, and R. J. Samulski. "Delivering transgenic DNA exceeding the carrying capacity...comparative study of strains of Halobacterium halobium differing in pigmentation." Biochemical and biophysical ...Schnütgen, Frank, et al. "A directional strategy for monitoring Cre-mediated recombination at the cellular level...

...make lentiviral vectors fantastic options for delivering shRNA, CRISPR/Cas9 components, and fluorescent...lines but reach their limits when it comes to transferring DNA into primary cells or non-dividing cells...virus. Even though this system was efficient at transferring the gene of interest to the appropriate cells...

...pipetting! We’ll release a new tip every two weeks during the school year. Our first quick tip, “Your Bench...  Want your quick tips even quicker? We’ll be sharing our favorite parts of every quick tip video on ...

...collection of memorabilia, even made an appearance during co-founder and CSO Melina Fan's words of welcome...you all so much for an amazing twenty years of sharing science!       ...

... What a fun video of Mario, dressed as Santa, delivering gifts to the Mushroom Kingdom. Kravitz Lab 2019...snowflakes, ornaments, lights, and took a group photo wearing holiday sweaters and Santa hats. There was even...

...Bacterial Expression Systems. Check out our Genome Engineering Overview and our CRISPR guide to find out how...how CRISPR is being used for bacterial genome engineering. To make it easier for you to find plasmids ...

...plasmid-based technologies. Commentary about scientific sharing and the open-access movement. At Addgene, we believe...the conversation! Do you have a question about culturing iPS cells? Confused by which system to use for...

...and to get some insight into her management and mentoring styles. If you'd like to learn more about Professor...defect in a single gene) 26:00-28:39 : Methods for Delivering Multiple Genes to Cells for Gene Therapy 28:40...

...standardized way. Since Addgene encourages the public sharing of aggregated data only, information on requesting...you find the dashboard useful when analyzing and sharing your own depositor metrics!  About the Author Joachim...

...packaged on request, you’ll see a banner below the ordering table. Click on it, select your desired serotype...find this service useful in your research. Happy vectoring! ...

...vectors repository since March.  Addgene is now offering an AAV2 CAG-driven tTA expresser from the Gradinaru...PdCO, deposited by the Yizhar lab. Addgene is now offering AAV1 and AAV5 preparations of several variants...

...experiences as well as plasmids from Addgene to bring new opportunities to Ghanaian scientists Click ...synthetic biology, DNA assembly methods, and engineering cellulose degradation. 18:21 - 23:26 : Kwabena's...

...direct impact on human health and look forward to hearing from her in the future. Future awardees We will...to learn more about future awardees and their inspiring research. Additional Resources on the Addgene...

...Intern (and soon to be Addgene Scientist!) Mary Gearing, this post focuses on new data from the Kim and...concrete context. I recommend this post to anyone wondering if and how they can apply FRET in their own research...