...lentivirus, AAV does not possess the ability to replicate itself and depends on helper viruses like adenovirus...herpes simplex virus to facilitate efficient replication. The most common production system, however, ...RNA genes; and the pRepCap plasmid containing replication factors and serotype-specific capsid genes.  ... steps of AAV production, iodixanol gradient purification, and AAV titration by qPCR.   Pro tip! An optional...al., 2010). For certain in vitro and in vivo applications, you can consider using unpurified AAV from ...Benyamini et al., 2023) to save time and cost.  Purification To purify your rAAV from your supernatant, you...recommend using a Tris-based lysis buffer and sonication.  The obtained virions from cells and supernatant...

...easier of the two, but it actually requires more technical skill to place the needle in the correct vein ...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: ...between the layers of the skin. While this could theoretically be performed anywhere on the body, somewhere... correct location requires a lot of skill and anatomical knowledge. Intrathecal injections are used for...stomach. This technique involves administering medication through a feeding tube with a bulb on the end...

...CRISPR mammalian expression systems, the typical applications for each, and potential delivery methods...well as suitable applications. Expression System Components of System Application Mammalian expression...Select the appropriate CRISPR tools for your application: Wildtype Cas9 or Cas9 nickase are appropriate... or introducing mutations and tags, while a catalytically dead dCas9 can be used in conjunction with activator...or more sgRNAs and Cas9 are required for any application. If you want to include a homology directed repair... be constitutive or inducible. U6 promoter is typically used for gRNA. May contain reporter gene (e.g....key features of common  delivery methods below: Chemical transfection   Lipid mediated Cationic polymers...

...Lee and Pantelis Tsoulfas of the Department of Neurological Surgery at the University of Miami. The beginning...expressing eGFP under the human ubiquitin C promoter. Anatomical abbreviations: Gr= Gracile tract, Cu= Cuneate...originally named orthogonal-plane fluorescence optical sectioning (OPFOS) (2). Arne Voie, David Burns ...of LSFM microscopes (3,4). After this initial application, Stelzer’s group described the single-plane or.... confocal microscopy In confocal microscopes, optical sectioning of a specimen is based on discriminating...used in LSFM. In LSFM the laser light sheet, typically 2-6  microns, illuminates only one thin plane ... milliseconds to acquire one image, thereby significantly reducing the time it takes to image through ...

... to be crazy complicated! Listen to the full podcast for more great science communication tips or listen...tons of ways you can get involved in science communication. In this second half of our conversation with...many things you can do start your own science communication student group and get more involved with your...is a graduate student group with the goal of communicating science to the general public and training the... the next generation of science communicators. In this chapter we learn about some of their events - science... peer-to-peer feedback in effective science communication. Chapter 7: The Goal of the SITN Podcast - Vini...Pint" event is the best way to get a Science Communication group started. It's as simple as getting people...

...transformation. A given insert will typically contain both a protein modification sequence and a selectable gene...nearly all domains of life repair genomic damage, specifically double strand breaks. Researchers have long ...tags have been integrated. Simply design your amplification primers with the desired targeting homology—... green, and red FPs that are codon optimized specifically for expression in yeast. These tagging vectors...This collection is ideal for tandem affinity purification of protein complexes. John Pringle and Jürg ...deposited a large collection of plasmids for genome modification in yeast developed by Dr. Pringle’s former lab...collection of plasmids for a wide variety of genome modifications in S. Pombe, including full and partial gene...

... plasmid or group of plasmids' functions and applications. We hope that these articles make it easier ...manipulating interneurons using a viral approach Genetically modified organisms like transgenic mice are important...FLIM (Fluorescence Lifetime Imaging Microscopy) applications. To demonstrate the power of mCyFP1, the Lin... cell physiology and molecular processes via genetically encoded proteins. PhoCl is a new member of this...experiment, check out our recent guest blog series: A Practical Approach to Choosing the B(right)est Fluorescent... Coselection efficiency is so high that some applications may permit working with edited “pools” of cells...showed high editing efficiencies with AsCpf1, indicating that ouabain coselection is a robust and flexible...

...backgrounds with technical expertise that ranges from astronomy to bioethics to chemical engineering and...Citizenship Initiative.    Get practical experience in science communication by writing for the Journal of...societal pressures including economic, cultural, and political. Science policy looks differently depending on... policy implementation, program management, communications, diplomacy and international relations, advocacy... p” policy.  “Big P” policy is what you might typically think of. It occurs in a government setting: drafting...STPF cohort members came to DC to do either a sabbatical year or transition away from academia as tenured...move into science policy. Read broadly about the historical roots of science policy to connect to your why...

...club occurs with a small technical team (single lab or team), a deep technical dive on single papers could...emerging work by collaboratively discussing recent publications in a specific field(s). Most anyone who has ...the company at large, by polling and informal communications we made a decision to try out a Bioinformatics...topic, there are a number of administrative and practical decisions to make. How often will you hold it?... the time recurrently carved out. This notice typically should include the paper, any supplemental data...repositories, or other information needed by someone to critically discuss the paper.  Before the first journal ...major findings and then focusing more on broader implications rather than diving deep into the individual ...

...culprit for these unusual cases was either 1) a physiological effect of SARS-CoV-2 toward destabilizing one...Adenovirus (and/or to its partner, AAV) or 2) a sociological effect of the COVID-19 lockdowns causing a lack...transcription) is demarcated by viral genome replication (Harrach, 2014). Given that the early genes ...encapsidate new virions) are not expressed. Note that replication-competent AdV vectors also exist and are used...the final topic, which is that natural AdVs, replication-competent viruses with a recombinant genome, ...basic research, like other viral vectors, though significant challenges remain (Wold & Toth, 2013).  While...Hermiston, T. W. (2003). Multigene expression from a replicating adenovirus using native viral promoters. Mol ...

... general public. Chemical Society in University College Dublin, Ireland The Chemical Society was founded...way up to professors. Oxford Biological Society, England The Biological Society based at the University...idea of the Freshers Fair and some of the great biological societies that Addgene has had the opportunity...at Freshers Fairs in the past year. Cambridge Biological Society, England Since 1996, Cambridge Biosoc...Biosoc’s goal has been to promote interest in the biological sciences amongst the student community at the...sessions to help undergraduate students with their applications. The society currently has 1,283 members, who...At Addgene, we’re incredibly glad to see these biological societies engaging with young scholars. We believe...

...over a month ago I finished up my PhD in Biological and Biomedical Sciences at Harvard University and entered...talking to people about and communicating science. The path to science communication This may come as somewhat...researchers gain doing PhDs (i.e. technical writing, teaching, presenting, analytical thinking, time management... I would ultimately want to go into science communication but thought that I would be best poised to share...PhD myself. To continue to foster my science communication skills, I took on TA positions, tutored fifth...largely my work developing myself as a science communicator that got me here! Thus far being an Outreach... one of my supervisors warned me, the swift gratification of publishing posts can be addicting. Even more...

... depends on the biological question, and zebrafish are not ideal for every application. Disadvantages ...have been used since the 1930’s in a range of biological studies, including investigations into environmental... are powerful resources for investigating any biological process. High quality genome sequence A high-...mutant, which regulates a developmental pathway implicated in congenital heart defects and cancer. These...thousands of mutations in genes affecting diverse biological processes—not just development—are available ...DNA into the genome is useful for a variety of applications. For example, specific tissues such as blood...pathways control this process. Additional zebrafish applications and conclusions Zebrafish are also popular outside...

...the Janelia Research Campus of the Howard Hughes Medical Institute. Are you currently on or planning to .... Given this large sum, one might expect that applicants would work for months to hone a chalk talk. Yet...research vision is your approach to resolving a significant knowledge gap in your field or even creating ...rejection of a model and no clear alternatives. Typically, a research vision is broken up into three aims... basis of a single grant. Either way, think realistically about the time and resources required to feasibly... aims in more depth in the remaining time. The typical chalk talk structure is: What is the big question...field, and interesting. For example, “there is a significant need for healthy livers for transplants. Growth...

..., Kyung-Mi, et al. “Aptamers and Their Biological Applications.” Sensors (Basel). 2012; 12(1): 612–631...amplifying RNA sequences that could bind very specifically to target molecules. Using a technique called... Aptamer biosensors are used in a variety of applications, including detection of environmental pollutants... opens aptamers for use in many entirely new applications including aptamer based fluorescent biosensors... render this approach less sensitive in most applications. Learn About Fluorescent Biosensors Light-up..., MG proved to be too toxic for many in vivo applications, but this work proved the concept that RNA could...and biochips. Ultimately, there are just some applications that lend themselves to aptamer-based instead...

...design and its potential for clinical applications. Specifically, efficacy rates of Cas7-11 were reported...enzyme can lead to off targets and result in significant, negative effects in RNA targeting experiments...originally suspected that this new Cas7-11 might be phylogenetically classified as the more complex Class I systems...non-specific activity. Its major advantage is the significantly reduced amount of off-target toxicity compared...using Prime editing alongside some creative (and significant!) protein engineering. They combined Prime editing...place, while increasing the number of potential applications. But they weren’t done engineering quite yet...switched up the serine integrase for one which can enzymatically take a genetic sequence and integrate it into...

...formal scientific communications, they are fantastic fodder for science communication. The trick is to ... is far from the only way to communication science. Science communicators also use the hero’s journey,...this small glimpse into the world of science communication and can use what you've learned to share your...Finding Conflict in Science  In all science communication, what you are doing is telling a (nonfiction...details often aren’t included in scientific communications, but they’re firmly part of the story to the...conflict gives you an easy framework with which to communicate your science. The act of building towards your...your results right at the beginning of your communication, use conflict to get your audience invested ...

...up. Applications of photoactivatable fluorescent proteins Some of the most important applications for ... Shai, et al. "Photoactivatable genetically encoded calcium indicators for targeted neuronal imaging."...reversible photoactivation - and have enabled sophisticated imaging techniques.     Irreversible photoactivation...level of background fluorescence. For any PA-FP application, the brighter the fluorescence the better. More...photoactivation. A high contrast ratio usually indicates that a PA-FP has a low level of spontaneous fluorescence...for PA-FPs involve enhancing optical live imaging of proteins, organelles, and cells. Similar to FPs, PA-FPs...photostable green and red states. Beyond these imaging applications PA-FPs have been developed for use in yeast,...

...protein composition. It is used for a variety of applications such as measuring target protein expression ...expression levels, assessing post-translational modifications, determining cell health, analyzing cell cycle stages...of the population as a whole, scientists gain statistical power in their observations. However, while flow...in single-file where it is interrogated by the optical system (Figure 1).   Figure 1: Cells expressing...complexity, measured using visible light scatter The optical system has both visible and fluorescent light sources...cell expressing low levels of the protein. The electrical system converts the fluorescence intensity to...intensity events are assigned to higher channels. In a typical assay a user will compare the shifting intensity...

...study its function is one of the most popular applications of fluorescent proteins. These fusion proteins... from Aequorea victoria and its variants are genetically encoded fluorescent probes. One of the limitations...motifs that are necessary for post-translational modifications. Despite these potential issues, GFP has been...ultracentrifugation or gel filtration is the most common analytical method. However, these may not reflect the situation...situation in cells. Therefore, the localization of critical fusions (e.g. tubulin or connexin) has been used...linker sequence short In the early days of GFP applications, many were concerned with steric hindrance by...fluorescent protein and protein of interest (POI). To indicate the sequence, people often use terminology like...

...components of the cytoskeleton. They provide cells with physical structure, separate chromosomes during mitosis...filopodia formation, cell contraction, or other morphological changes, depending on the specific Rho GTPase...RhoGEF is a great way to stimulate and study these physical processes. Joachim Goedhart and colleagues in ... binding of the plasma membrane. In previous publications, they described tools to control formation of...the effect to a specific time or location can be critical when, say, the gene is essential during development...isn’t powerful enough, what about altering the chemical composition of the membrane itself? The Baskin...Baskin Lab previously published OptoPLD, an optogenetically controlled phospholipase D (PLD). Now they present...

...cryogenic electron microscopy (cryo-EM) protocols typically require high concentration and purity of the target... (Figure 1). This enrichment streamlines the purification and minimizes sample loss while also supporting...chaperone NPM2, but the technique should be widely applicable to other targets. The modular system of linkers...scFvs for other targets or could be used for applications beyond cryo-EM.   Find plasmids for MagIC-Cryo-EM...interact with the air-water interface (AWI) before vitrification, they can be completely denaturated (left) or...right). B) Protein samples protected from AWI by helical LEAs. C-E) cryo-EM single-particle analysis of ...orientations, this could be overcome with methods such as chemical crosslinking or tilted-stage imaging. A truncated...

...people who are data oriented, scientifically minded, and able to communicate complex information - all traits... incredibly important to the stability of the biological research enterprise going forward, in this post...the FOR website, the FOR Boston 2015 organizers indicate that the main question they wanted to focus on...Publishing Panel brought in editors from a variety of publications as well as young PIs and all gave advice on ...blogging, write reviews, or write for a university publication, improving your general writing skills will help...academic publishing goals and make you a better communicator overall. Don’t Write in a Vacuum Your research...cognizant of this during my PhD. Submitting a publication doesn’t have to be like throwing it into a black...

...non-viral methods including both chemical and physical methods in the lab. Chemicals like calcium phosphate and...Scientists can also use electroporation, the application of an electrical charge to cells, to increase cell membrane...take up DNA, they can be made competent through chemical manipulation in the lab. This is commonly done... properties. The phagemids are packaged into replication-incompetent phage particles with assistance from... found on an episome, a piece of DNA that can replicate on its own or be integrated within a bacterial...easily cross the negatively charged cell membrane. Physical methods such as electroporation or microinjection...cells. Electroporation on the other hand uses electrical pulses to create transient pores in the cell ...

...potential applications. Challenges of existing mutagenesis methods Several continuous diversification methods... challenging for users to achieve targeted diversification of all nucleotides within a defined region ...Chang Lab have created methods that introduce diversification by targeting a site of interest with dCas9 ...level of variation that can be introduced.   Diversification is limited due to the integration of oligonucleotide...increasing the standard mutation rate seen during DNA replication by 120-fold over background. Due to its modular...only ~2-fold over nCas9 levels. Two sets of modifications were also made to PolI3M to increase the mutation...more than one mutation near the target site. Applications of EvolvR The Dueber Lab used EvolvR to discover...

...tools from across the biological spectrum to learn more about the brain and neurological disease. I was surprised...and, like many biological fields the number potential of experiments enabled by physical, software, and...the things that popped out to me as a novice and indicate why other neuroscience newbies might want to get...brain functions and vice versa. Even within the biological sciences, there’s plenty of interaction between... microbial DNA they all seem to require some mathematical know-how for analysis. If you’re looking to ...presentation area, I honed in on projects using biological tools. In addition to the researcher using sequencing...but are afraid you don’t have the right kind of biological expertise to get into the field, there’s likely...

... given prokaryote typically has genes encoding one or a few restriction modification systems containing...methylation sites are not specifically associated with any restriction modification systems, their sequences... one half of naturally occurring restriction modification systems that prokaryotes use to protect themselves... phage for instance). Aside from restriction modification systems, DNA methylation also plays an integral...integral role in regulating genome replication, repairing mismatched basepairs or small indels that occur...methylases) are independent from the restriction modification systems, yet can still affect whether certain... These specialized E. coli strains have been specifically engineered to be Dam and Dcm methylase-deficient...

...suggesting that QKI is a host protein critical to Zika replication. Human genetic disease. RaPID can also...methods Not in a native context. While native purification preserves RNA structure and naturally occurring...present in the cell, it can also introduce non-physiological RNA-protein interactions due to sample processing...t be used to study endogenous RNAs at their physiological expression levels; however, knock-in of BoxB...used the Contamination Repository for Affinity Purification (CRAPome), to identify highly biotinylated background...research groups that performed BirA*-based affinity purification mass spec experiments. These background proteins...subtracted from Ramanathan et al.’s RaPID datasets. Applications of RaPID RaPID sounds like a nifty technique...

...of DNA that can potentially complicate results of a downstream application such as quantitative PCR to...sulfate on the activity of ribonuclease A. Biochimica et Biophysica Acta (BBA) - Enzymology 341:484–488. https...easier. Recently, we published a blog on DNA purification without a kit that outlined several reasons ...take care whenever doing any type of DNA or RNA purification to avoid contamination, take extra care when..., that isn’t the case for RNA extraction and purification. There is one simple method that works, and ... within cells or tissues- they don’t actually chemically stabilize the RNA molecules (Allewell and Sarma... MacDonald RJ, Rutter WJ (1979) Isolation of biologically active ribonucleic acid from sources enriched...

...regulation thats as sophisticated as a natural network. We could build a material as sophisticated as wood. To...to be able to design new materials and chemicals as sophisticated as those that living cells and animals...Technology has been “working on new experimental and theoretical methods to push the scale of genetic engineering...for major advances in agriculture, materials, chemicals, and medicine. As they’ve gone along, Voigt’s ... make a very large number of regulators. That basically means larger genetic circuits that could conceivably... perspective and you can layer them for more complicated functions. Those things are easy with CRISPR.... carbons and oxygens. Nothing too much more sophisticated. There are also proteins, like antibodies. But...