...have proposed the concurrent use of “orthogonal” Cas9 proteins derived from different bacterial species...variants—nuclease-competent or nuclease-null—in order to effect different perturbations. Consider, for instance, the challenge... both cutting and regulation of expression at different genetic loci within a single cell. One might imagine...expression (6). Altering the length of gRNAs targeting different sites of interest thus represents a straightforward...introduced together with truncated gRNAs targeting a different set of loci to induce simultaneous cutting and...DNA sequence or regulating gene expression.     References 1. Esvelt, Kevin M., et al. "Orthogonal Cas9 ...

...into the margins of my protocol. While these tips weren’t critical to the experiment, they definitely made... AAV packaging plasmids. The cells need three different plasmids to produce AAV: 1) the RepCap plasmid...gradient ultracentrifugation uses a gradient of different concentrations of iodixanol to separate out contaminants...titer generated by qPCR is useful, I test a few different concentrations of virus, or multiplicity of infections...variability. Check out this post to learn more about different ways to titer AAV. Do you have any AAV production...teaching me about AAV production and purification! References 1. Aurnhammer, C., Haase, M., Muether, N., Hausl...adeno-associated viral vectors for in vitro and in vivo use. Current protocols in molecular biology, Chapter 16, Unit...

...safely and transparently.  For more information, visit www.sculptingevolution.org. References: Burt, ...didn't work quite as well as anticipated4, greater awareness will certainly improve safety. What safeguards...vectors of diseases such as malaria and dengue are currently being performed in regions that don't have resident...Cas9 genome editing. Finally, gene drives are an inherently collective technology: because they necessarily...dengue for many years. Since more than a thousand children died of malaria today, let's not risk a potential...information about gene drives can be found in the referenced publications. Thank you to our guest blogger...

... mutagenesis or gene targeting Victor de Lorenzo's lab has engineered a modular mini-Tn5 vector that... microscope 8 hours post-pulse. The distinct differences of red-to-green fluorescence in individual mitochondria...mitochondria in the networks marks the differences in import of newly synthesized (green) protein and ...five previously unknown channelrhodopsins from different species: Stigleoclonium helveticum (ShChR), Chlamydomonas...cassette. They validated this methodology in two differents types of bacteria (E. coli and Salmonella typhimurium... of all four final RVD modules in each of two different TALE scaffolds are provided, as the optimal scaffold...region of one neuron and postsynaptic region of a different neuron, come into proximity in the synaptic cleft...

...delivered on a different plasmid and allows simultaneous multiplex editing of six different genes. This ...flanking the promoter-gRNA construct, but with different sequences adjacent to the sites. When digested...assembly principles, but they’re optimized for different purposes.   Gersbach Lab multiplexing plasmids...kanamycin-resistant plasmids, each containing a different gRNA target sequence downstream of the 7SK, human...users to express up to 7 gRNAs. The kit contains different destination vectors depending on the total number...transcript. Rather than being transcribed from different promoters, the gRNAs are transcribed together ...likely be used in a wide range of bacteria. References 1. Maddalo, Danilo, et al. “In vivo engineering...

...It was by serendipity that I got into the field of gene therapy, more specifically AAV-based retinal ...treatment, famously visited Congress to help increase awareness about the potential of gene therapy!). It would...is available. While only natural serotypes are currently used in clinical trials, a variety of engineered... AAV safety profile is excellent and further strengthens its position as the vector of choice for in vivo...and the development of personalized medicine. References and further reading 1. FDA approves gene therapy...the Light after 25 Years of Retinal Gene Therapy. Trends Mol Med. 2018, S1471-4914(18)30123-0. DOI: 10.1016... Karen Guerin...

...space to be oxidized and form disulfide bonds (Frenzel et. al., 2013). If you’re using a mammalian production...: The steps of rAb production. Created with biorender.com   Another method of harvesting uses magnetic...useful to compare recombinant antibodies to their parental hybridomas. Aggregation and storage Aggregation...our Antibodies 101 blog series.  Resources and references More resources on the Addgene blog Introduction...Purification Protocol Addgene's Antibody Guide References Trimmer JS. Recombinant Antibodies in Basic Neuroscience...: 10.1007/978-1-4939-8730-6_1. PMID: 30242676. Frenzel A, Hust M, Schirrmann T. Expression of recombinant...particle formation, characterization & rheology, Current Opinion in Colloid & Interface Science, Volume ...

...Testing of three different AAV serotypes (AAV1, 8, 9) injected into the A2 noradrenergic area of the caudal...helping everybody, helps everybody! References and resources References Dayton, R. D., Grames, M. S., & ...scientists under-informed with respect to how different serotypes, promoters, and injection methods are...literature, scientists can use the Data Hub to compare different methods and vectors, and use this data to guide...particle aggregation and methods to prevent its occurrence during vector purification and formulation. Molecular...

... as fixing and permeabilizing.  References and resources References Brown-Harding H (2020) Fixation artifacts...previously hidden within the protein. Created with BioRender.   Permeabilization What is the point?  Antibodies...100, Tween-20, or saponin. Each detergent has different chemical properties that will impact the level...concentration.      Figure 2: Examples of different permeabilization strategies and when to use them...permeabilizing organellar membranes. Created with BioRender.   How do I choose?! Ok, so now you know some...https://doi.org/10.1177/33.8.3894499    Im K, Mareninov S, Diaz MFP, Yong WH (2018) An Introduction to...

...be identified by PCR later. The PNS presents a different problem: it’s spread throughout the body and is..., AAV.PHP.eB was used at a lower dose than it’s parent AAV.PHP.B: 1x1011 vs 1x1012 viral genomes (vg) ...comparison of AAV.PHP.eB and AAV.PHP.S to their parental vectors. To quantify this comparison, capsids ...efficiency of AAV-PHP.eB and AAV-PHP.S versus their parental AAV vectors. What can these AAV’s be used for...   Cortex Striatum Cerebellum AAV.PHP.B (parental) 49 +/- 7% 36 +/- 8% 37 +/- 10% AAV.PHP.eB 69...Transduced   Dorsal Root Ganglion (DRG) AAV.9 (parental) 46 +/- 0.7% AAV.PHP.S 82 +/- 2% Table 2:... and supplemental figure 8 in Chan et al).  References 1. Chan, KY, Jang MJ, Yoo BB, et al. Engineered...

...colleagues reported that it is possible to reprogram differentiated cells into induced pluripotent stem cells (...benefit when recruiting patients, specifically children and patients with skin disorders for research ...OriP and induces amplification of the episomes concurrently with DNA amplification in the host cell. This...integration-free, karyotypically normal and are able to differentiate into various cell types of all three germ layers...Kushi) Mukherjee. Kusumika (Kushi) Mukherjee is currently a postdoctoral fellow at Massachusetts General...https://www.linkedin.com/in/kmukherjeephd/.     References 1. El Hokayem, Jimmy, Holly N. Cukier, and Derek...Interview with Darrell Kotton Four Factors that Differentiate the Stem Cell Field Sign Up to Write for the...

...can be used with or as fluorophores in several different ways. Researchers have used oligonucleotides in...were born. Aptabeacons can be created in many different ways, but the general premise is that an aptamer...relying on a lack of fluorescence for readout may render this approach less sensitive in most applications.... As a product of SELEX and FACS, Broccoli is inherently more stable in cells. The new aptamer also has...stable and can go through cycles of denaturation/renaturation. In vivo assays in which protein fluors are...Aptamers can be selected for these small ligands. References 1. Ma, Hanhui, et al. “Multicolor CRISPR labeling...Shiping, et al. “Aptamer-based biosensors.” TrAC Trends in Analytical Chemistry 27(2):108-117 · February...

...per video abstract at 2,947 views. The journals Current Biology (2,500 views), Journal of Number Theory... Addgene Youtube Channel   After watching these trends for a few years, I decided to look at what online...examples for creating effective research videos.  Karen McKee, also known as the science videographer, is...access online journals or even use a computer.  Currently, two of the more popular biology videos are:   ...published in a recent issue of the open access journal Current Biology. Video on We Share Science: http://wesharescience.com...wesharescience.com/pin/7472 Article: http://www.cell.com/current-biology/abstract/S0960-9822(16)30873-9 Youtube:...WeShareScience or email Ryan at [email protected]   References 1. Larivière, Vincent, Yves Gingras, and Éric...

... to produce abundant, externally fertilized, transparent progeny (Figure 2). These features give it a ...individual to maintain. Also in contrast to mice, the transparent zebrafish embryo develops outside of the mother...researchers to observe changes to these tissues under different experimental conditions. Strategies for generating...sequencing approaches (Harland, 2018). Several different methods have been developed, relying on established...zebrafish—including the available genetic resources and transparency of the embryo—have proven useful in a variety...vertebratedevelopment using optogenetic approaches.     References El-Brolosy, Mohamed A., et al. "Genetic compensation...plasmid collection Read our molecular biology reference Find plasmids for your research ...

...scientists have since adapted it for a host of different proteins and small molecules from a variety of...needs. Herein, we’ll give you a rundown of the different types of ELISA, advantages and disadvantages of...in the sample. This figure was created with BioRender.com  Direct ELISAs A direct ELISA is the quickest...this method, the target antigen competes with a reference antigen for binding to a primary antibody.  While...typically users first coat the multiwell plate with a reference antigen and then they preincubate the antigen-...allow the unbound primary antibody to bind to the reference antigen. The more antigen in the sample, the more... means less antibody available to bind to the reference antigen, producing a weaker signal.  The major...

...Emily P. Bentley in December 2024. References and Resources References Anzalone, A. V., Gao, X. D., Podracky...(Anzalone et al., 2019). Plus, it can combine different types of edits with one another. All of this is... An overview of prime editing. Created with BioRender.com. How does it work? First, an engineered ...precise than base editing (in certain circumstances) Current base editors can perform any base-to-base conversion...indels than prime editing. But for positions that aren’t well positioned within the editing window, prime...type, and goal of the experiment can all impose different constraints and affect how you select and design...5.1-fold (though up to 45-fold) higher across different genomic sites compared to PE1 (Anzalone et al....

...you to do many different molecular biology techniques (such as fusing it with different tags, putting ...Addgene's reference guide on molecular cloning techniques, including information on seven commonly used... Molecular Biology Reference Choosing a Molecular Cloning Technique Molecular Cloning...availability of starting materials or just personal preference. Check out our blog on choosing the right cloning...Gateway® destination plasmids have been made with different promoters, tags and fluorescent proteins. Thus... variety of promoters and into backbones with different selection cassettes). Although gateway cloning...base pairing in the annealed product, ligation is rendered unnecessary. The product may be transformed directly...

...research coming out of the lab.  References and resources References Hao, Y., Yan, J., Fraser, C., Jiang...affinity. This extended-open state is key to strengthening ligand binding, mediating cell adhesion and ...then used these antibodies to show the binding preferences of the αV subunit and its β partners. Of interest...antibodies and affinity reagents to study the difference in cell morphology changes induced by integrins...antibodies comes from IPI’s work with the Boston Children’s Hospital and Harvard-based lab of Tim Springer...discovered by yeast display reveal αV subunit pairing preferences with β subunits (p. 2024.01.26.577394). bioRxiv...

...disease postnatally. Now, three papers published concurrently in Science have shown CRISPR can treat a genetic... Even low-frequency editing could make a huge difference in DMD, and it’s estimated that exon skipping...respectively. Although each study used slightly different methods, they each observed the same positive ...necrosis compared to unedited animals. In grip strength, specific force, and other muscle tests, muscle...disease type C. Although the three DMD studies referenced here represent a great step forward for CRISPR...many distinct CRISPR therapies to match these different mutations. For each therapy, effectiveness and...treatments may also be closer than we might expect.   References 1. Kaiser J. CRISPR helps heal mice with muscular...

... you need. Most often, early career scientists aren’t trained to have these types of negotiations. They...all of the major equipment that you use in your current lab first. Generally, major equipment can be defined..., glassware, etc), get a simple list from your current P.I., lab manager, or administrator containing ... Do online searches, and seek advice from your current administrative services, managers, and techs’ financial... is where you can start to identify which items aren’t critical to your research success. If you can get...can still go horribly awry when several factors aren’t taken into consideration. 1. Always focus on the...for the success of your science. If the resources aren’t available, then you’re entering a win-lose situation...