...Many thanks to our guest blogger, Doug Richardson! Doug Richardson is the Director of the Harvard Center...This post was contributed by Doug Richardson, Director of the Harvard Center for Biological Imaging and...microscope designs may further exploit the unique characteristics of fluorophores to block out of focus fluorescence...blurry out-of-focus light. The most difficult characteristic to capture in these types of samples is their...model embryos >15 μm Thsee are some of the most challenging samples to image as they require both optical... 2, 932-940 (2005). PubMed PMID: 16299478. 9. Richardson DS, Lichtman JW. Clarifying Tissue Clearing. ...

...lack of caveolin 1 in breast cancer also induces changes of stromal cells in the microenvironment. This ...from cancer cells to the microenvironment. This mechanism happens for both short and long distances. When...transferred to other cells, inducing epigenetic changes in targeted cells. In this case, exosomes may transport...extracellular matrix and fibroblasts. With each cell mechanism discovered in microenvironments, we are one step...References Allinen, Minna, et al. "Molecular characterization of the tumor microenvironment in breast cancer...Intercellular nanovesicle‐mediated microRNA transfer: A mechanism of environmental modulation of hepatocellular ...

...and RNA expression - but what if you just want to change protein levels? Until recently, no such tool existed...proliferation. These cells also did not contain changes in expression of TRIM21-associated genes, indicating...the first author of this article, Dr. Douglas Richardson, recently contributed a relevant post to the ...Addgene blog. Find the SRpHi Biosensors here. Richardson et al. Nat Communications. 2017. PubMed PMID:...transcription factors in series and independently changes inducer levels. That is, if transcription factor...expression of a reporter protein, it is possible to change the amount of noise in reporter gene expression...

...in comparison to protein-coding genes (Rinn and Chang 2012). Scientists have determined the function ... Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A Programmable Dual-RNA–Guided DNA Endonuclease...CRISPR-Based Approaches for the High-Throughput Characterization of Long Non-Coding RNAs. Non-Coding RNA. 2021.... doi:10.3390/ncrna7040079 Horlbeck MA, Liu SJ, Chang HY, Lim DA, Weissman JS. Fitness effects of CRISPR...(5):573. doi:10.1038/s41587-020-0428-0 Rinn JL, Chang HY. Genome Regulation by Long Noncoding RNAs. Annual...in Plants with the CRISPR-Cas Technology is a Challenge yet Worth Accepting. Front Plant Sci. 2015;6. ...

...•T-to-G•C base changes (Figure 1), but it can be challenging to make these base changes, especially in...hemoglobin. Previous studies have shown that one base change in the promoter region of gamma hemoglobin (HGB1...effects. ABEs have been described to introduce base changes in cellular DNA and RNA dependent or independent...authors tested ABE8.17-m and found that one base change mutation (ABE8.17-m+V106W) (Gaudelli et al., 2020...

... heteroduplex is cradled in the positively charged channel between the two lobes. For CRISPR to be programmable...light blue; bridge helix, purple.   Apo Cas9 changes conformation frequently (Osuka et al., 2018), but...structure, reflecting the less stringent recognition mechanism that can tolerate mismatches in this region. The...in the duplex that may not fit properly in this channel, depending on their precise location. The end of... BioRender.com.   The exact conformational changes that make Cas9 such a valuable laboratory tool ...Doudna, J. A. (2017). CRISPR-Cas9 Structures and Mechanisms. Annual Review of Biophysics, 46, 505–529. https...

...institutional oversight is required. Restraint: mechanical restraint or anesthesia Location: tail vein or...you might after getting a vaccine. Restraint: mechanical restraint Location: thigh Gauge: 25–27 Volume...xenograft models. Restraint: back scruffing or mechanical restraint Location: back or hind legs Gauge: ...Restraint Location Gauge Volume Skill Intravenous Mechanical restrainer or anesthesia Tail vein or retro-orbital...cavity 25–27 < 2–3 mL Intermediate Intramuscular Mechanical restrainer Thigh 25–27 < 0.02–0.05 mL Beginner...Beginner Subcutaneous Back scruffing or mechanical restrainer Back or hind legs; between skin and muscle...

...cancerous cells Luckily, there are several safety mechanisms in place to protect us from rogue cells when ...programmed cell death, or apoptosis, is one such mechanism. When a cell or its genetic material is damaged...daily, yet obstacles remain. One of the biggest challenges facing researchers today is the complexity of... of the latest resources? In response to this challenge, Addgene has launched an initiative to curate ... your thoughts in the comments section below. Michael G. Lemieux is a Scientist at Addgene with interests... Michael G. Lemieux...

...Did you notice the “Filter By…” options changed when we changed from searching for viral preps instead ...best plasmid for your research can be a daunting challenge. With over 80,000 deposited plasmids from around...assembled a series of screenshots to highlight changes to both the design and functionality of searches...of plasmids? The available filtering options now change dynamically based on the type of material and specific...you may find the available categories and filters change as we now only show options relevant to your current...

...Disable the other repair mechanisms Mammalian cells have 2 primary repair mechanisms besides HR - Non-homologous...mammalian cells, but a less frequent DNA repair mechanism in general. On average, it accounts for less than...is a silent mutation, so you don’t accidentally change your protein of interest.   Fig. 2: Stop...potentially shuttling repair down a different pathway (Charpentier et al., Nat Com).     Fig. 3: HDR events... 8(2024), (2017). 10.1038/s41467-017-01836-2 Charpentier, M., Khedher, A. H. Y., Menerot, S., Brion, A...

...again and possibly mediate HDR. This “second chance” mechanism might improve the frequency of desired HDR...al. added to the CRISPR toolbox with their characterization of two Cpf1 orthologs that display cleavage...requirement. Zetsche et al. started from square one to characterize the Cpf1 nucleases. Using Francisella Cpf1 (...elements of CRISPR-Cpf1, Zetsche et al. turned to characterizing its cleavage pattern. Again, they were in for...efficiency. Kim et al. and Kleinstiver et al. characterized genome-wide editing efficiency of two Cpf1 orthologs...

...sensitivity Longer protocolIncreased chance of cross-reactivityChallenging to develop assay Competitive...antibodies. Assay development, however, can be challenging since scientists must first identify two independent...Disadvantages Direct Fast and simple protocolReduced chance of cross-reactivity Higher level of background...availability Longer protocolLess specificityIncreased chance of cross-reactivity Sandwich High specificityHigh...Competitive Appropriate for small moleculesReduced chance of cross-reactivity Less sensitivityLess specificityLimited...

...research. This includes chat apps like Slack, Microsoft Teams, and Google Hangouts Chat, resources like Github...an electronic environment and hence making it searchable and shareable, but some ELNs provide better support... delve deeper and look specifically at the characteristics and capabilities an ELN needs to facilitate...send documents from RSpace to a selected Slack channel for discussion and review. It’s also possible to...many different ELNs out there can help with searchability, record-keeping, and data sharing, not all of...

...on-site, but now we were presented with a new challenge: automating the bioinformatics steps needed to...input to the next via a Nextflow concept known as channels. Since a Nextflow process can utilize any scripting...process.  Next, we specified the input and output channels for the initial process and tested it by running...number of input file names or filtered the input channel to only use the first n items. To be sure, we went...first, to make sure we understood the concept of channels and implemented the input declaration correctly...faster runtime and decreased cost compared to purchasing and maintaining our own infrastructure. Our patchwork...

... LA, Garcia KF, Barnes KG, Chak B, Mondini A, Nogueira ML, Isern S, Michael SF, Lorenzana I, Yozwiak NL...reporter is added to the reaction. Cleavage of the quenchable fluorescent RNA by the “activated” Cas13 produces...Csm6 specific reporter (in the same fluorescent channel as the Cas13 reporter), they could significantly...Donghia N, Daringer NM, Freije CA, Myhrvold C, Bhattacharyya RP, Livny J, Regev A, Koonin EV, Hung DT, Sabeti... Koonin EV (2015) Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems. Molecular...

...may be familiar with popular light-sensitive ion channels used to control activities like neuronal signaling...activity. Researchers have designed a variety of photoswitchable elements into many other types of proteins....under blue light BcLOV4 undergoes a conformational change and binds the plasma membrane under blue light ...The authors demonstrated its use to probe cell mechanics, vesicle transport, and cell division, but given...family signaling pathways. Rho guanine-nucleotide exchange factors (GEFs) activate their partner Rho GTPase...formation, cell contraction, or other morphological changes, depending on the specific Rho GTPase/GEF pair ...

... of the fly (Pfeiffer et al., 2010). Yeast (Saccharomyces cerevisiae) Yeast, one of the simplest eukaryotic...as Parkinson’s disease (PD). PD is primarily characterized by protein misfolding that leads to a build-...invaluable in understanding the basic molecular mechanisms underlying the disease (Menezes et al., 2015)... development, cell signaling, aging, and mechanotransduction to name a few. For example, Andrew Leifer....5 (2011): 190-195. Liu, Wei, et al. "From Saccharomyces cerevisiae to human: The important gene co‑expression... PubMed PMID: 26712014. Pandey, Udai Bhan, and Charles D. Nichols. "Human disease models in Drosophila...

...Stochastic multicolor labeling is a popular technique in neuroscience and developmental biology. This... persistently fluorescent during development. Stochastic multicolor labeling systems, many based on Brainbow...even axons of up to 100 microns in length. Some challenges of the Brainbow system are that it does not use...for improving the level of detail possible for stochastic multicolor labeling of the brain. Labeling Drosophila... researchers who developed Tetbow. Conclusion Stochastic multicolor cell labeling systems allow single...Harbor Protocols 2011.1 (2011): pdb-prot5546. Richardson, Alexander, et al. "Visualizing the Fate of Transplanted...

...: In one of my favorite books, Michael Ende’s Momo, the title character endears herself to the nearby ...lack thereof): Information sharing gets more challenging the bigger an organization gets, and creating...communication that gets sent out through multiple channels is not reasonable or sustainable. That said, when...village (and to the reader) by simply listening. Chances are, you know someone like this, even if you’re...these people yourself.  Fig 1: Quote from Michael Ende, courtesy of morefamousquotes.com A good...

...to simultaneously test how multiple amino acid changes affect a protein of interest’s function. This technique...combination and possibly discover unexpected epistatic changes in protein activity. See Natarajan et al for an...mutagenic primers to introduce all desired codon changes (step 1 in the image above). A second universal...This results in the final product having a codon change present on both strands of the plasmid. The ssDNA...in your research? References 1. Natarajan, Chandrasekhar, et al. "Epistasis among adaptive mutations ... Mccullum, E. O., Williams, B. A., Zhang, J., & Chaput, J. C. (2010). “Random Mutagenesis by Error-Prone...