...establishment of two cell lines from the insectspodoptera frugiperda (lepidoptera; noctuidae). In Vitro, 13(4), ...packaging plasmids (three packaging plasmids for 3rd generation lentivirus production). The packaging plasmids...Insect cells are a less common option for the generation of viruses, though they are a popular choice ...from the pupa ovarian tissue of fall armyworms (Spodoptera frugiperda). Much like HEK-293T cells, they are...much larger packaging capacity — up to 38 kb! Generally, insect cell production methods produce lower ...versus insect cells for viral production Consideration Mammalian cells Insect cells Viruses AAV and...adeno-associated virus type 2 vectors. Human Gene Therapy, 13(16), 1935–43. https://doi.org/10.1089/10430340260355347...

... molecule). Degron tags can range in size from several kDas up to ~35 kDa and are typically knocked-in...manipulate protein expression. These tags offer several advantages over the alternative systems – CRISPR...interest to the proteasome for degradation. Like several other tags described here, the dTAG uses a small... and widespread usage, a proteolysis-targeting chimera (PROTAC) was developed for HaloTag. PROTACs are...their target protein and an E3 ligase through interacting ligands. After an E3 ligase facilitates ubiquitination... leakiness associated with the system, but the general concept of the system has remained the same. An...predict precisely how a given degron tag will cooperate with your protein of interest and how effective...

...signal you can generate without disturbing the integrity of the membrane, a concern with several of the previously...we will review how these indicators work, their general applications, and why viruses are essential to ...time from signal input to readable output, which generally exceeds that of the VSD GEVIs. However, since ... to the plasma membrane due to the addition of several targeting domains. The quencher and voltage detector...previously described systems. Several adaptations of this method have been to tag, cage, uncage, and trap...your experimental system.   Figure 2: General overview of chemigenetic tools. A genetically encoded...can be used for chemigenetics, biosensors, and generally as biological tools, our blog has you covered....

...have also been using Addgene CRISPR vectors to generate cell lines with knockouts of specific genes. Many...should be able to keep cells moist and hopefully aerated with a thin layer of molecular biology-grade agarose...sterile truncated pipet tips. Indeed, an experienced operator can pick 48 clones from a 10 cm dish in 15- 20...both Hepes and bicarbonate) is brought to room temperature and placed in the hood. The medium from a 10 ...is capped, swirled and tested for the correct temperature by placing on the wrist or cheek. Within less...the agarose cloning method Test your agarose temperature. Cloning cells using an agarose overlay does ...agarose onto the recipient plate at just the right temperature. If the agarose solution is too hot, the cells...

...by the Sternberg lab derives from Vibrio cholerae (V. cholerae INTEGRATE - hereafter VchINT) and encodes...and technical considerations Making insertions in bacterial genomes with the V. cholera INTEGRATE system... using Tn5), and as knock-in tools for human therapeutics (e.g. using Sleeping Beauty). Despite their ... CRISPR-Cas, these systems represent the next generation of tools for efficient and programmable insertions...achieved with VchINT, at two different incubation temperatures. Data adapted from Vo et al., 2020. In E...-recombineering, CRISPRa/i, and base editing. Leveraging a unique marriage between distinct biological...

... the Cas9 nuclease in the absence of genomic alterations. These studies utilize a catalytically inactive...dCas9 and targeting gRNA, to identify molecular interactions at this site (Fujita and Fujii, 2013). They ...method can characterize nucleic acid-nucleic acid interactions at a genomic locus (Fujita et al., 2017). CRISPR-mediated...Fujita et al., 2018). Furthermore, the Fujii Lab generated a transgenic mouse line and retroviral expression...when inappropriately expressed can cause facioscapulohumeral muscular dystrophy (FSHD), a disease for ...specific gRNA-dCas9 complex. When combined with next-generation sequencing, this method allows for the recovery...Toshitsugu, et al. "Identification of physical interactions between genomic regions by enCh IP‐Seq." Genes...

...and use a standard curve and provide some general considerations for scientists planning to use them in ...for the known concentrations (standard curve), generating an equation that allows you to determine the ... triplicate. The closeness of the data points generated by replicate values provides useful information.... With the help of graphing software, you then generate a trendline for the data. The appropriate trendline...users aim for an R2 above 0.95.    Additional considerations When running assays that rely on a standard...To calculate the %CV of the standard curve, one averages the individual %CVs for each point on the curve...assay. Try to minimize these.  Review previous literature or results from labmates to get a sense of the...

...broke down the method into six general steps and outlined the considerations to be made during each step....that occurs depends on the incubation time and temperature, so you may be able to recover good antibody ...fixation*. Organic Solvents Organic solvents, generally acetone, methanol, or ethanol, work by dehydrating...membrane integrity (Fig. 2). For example, saponin interacts with cholesterol in cell membranes and leaves ...Tween-20 are examples of non-ionic detergents that interact with lipids and proteins in membranes and permeabilize...to use your specific antibody for IF, look for general IF protocols for your sample type. Starting with...cross-linking fixation; they require an additional step generally referred to as Antigen Retrieval. I chose not ...

... and several others can be made fluorogenic using environmentally sensitive fluorophores. Overall, self-labeling...student searching couch cushions for spare change, desperate to extract a few more photons from a sample. Chemical...by Roger Tsien, who showed that the selective interaction between a bisarsenical dye ligand (e.g., FlAsH... lipoic acid ligase, biotin ligase, phosphopantetheinyltransferase) – These enzymes catalyze the covalent...corollary to these innovative labeling strategies, several groups—including mine—have been revisiting the ...PMID: 26000849. 2. Grimm, Jonathan B., et al. "A general method to improve fluorophores for live-cell and...Protein Should I Use? Resources on Addgene.org Generate Fluorescent Protein Fusions Request Plasmids from...

...After several decades of trying to address these challenges, researchers have amassed a considerable molecular... overexpress a recombinant protein? It is not generally in a cell’s best interest to overexpress a protein...peptide-based proteosome inhibitors, glutathione S-transferase (GST), which can be fused to recombinant proteins...for affinity and purification An affinity tag, generally a relatively small sequence of amino acids, is...Purification Binding and elution steps use very moderate buffer conditions FLAG DYKDDDD or DYKDDDDK or...interest would eventually get ridiculous (you generally don’t want more tag than protein), 2-3 tags is...IgG-binding domains. TAP has since come to encompass several other tag combinations, though frequently those...

...detected on commercial lateral flow strips, similar to pregnancy tests. With lateral flow strips, the presence... to cleave ssRNA or mRNA. It also exhibits a “collateral effect” of non-specific ssRNA cleavage. How does...sample is first amplified using RPA (recombinase polymerase amplification) or reverse transcriptase (RT)-... for nucleic acid detection almost anywhere as lateral flow strips are easy to transport and work rapidly...diagnostics (e.g. pathogen or virus detection), therapeutics and sensitive genotyping. The beauty of SHERLOCK...Cas12a however recognizes a different PAM site and generates 5’ and 3’ staggered ends after dsDNA breaks. The... DETECTR platform to detect SARS-CoV-2 using a lateral flow strip format. They published this in Nature...

...workflows considerably easier, faster, and more efficient than previous methods. Still, generating reliable...sequence you used. The ICE tool will calculate overall editing efficiency and determine the profiles of...detect or analyze complex edits, such as those generated by multiplexed delivery of multiple sgRNAs. Since...particular parameter was automatically adjusted to generate ICE results. If there are no results or there ... the ICE linear regression is computed during generation of the ICE Score, the Pearson correlation coefficient...around the cut site. It shows, base-by-base, the average amount of signal that disagrees with the reference...sequencing bases downstream of the cut. Summary ICE generates NGS-quality CRISPR editing analysis from Sanger...

...what online tools people generally like to use to share ideas – with the general goal of seeing if and how...their disciplines. Included in the article is an interactive chart where you can see the yearly interdisciplinarity...abstracts identified. The journal Cell had the largest average view count per video abstract at 2,947 views. The...Gastroenterology (1,046 views) rounded out the top four average video abstract view counts.” For researchers looking...offers a low-barrier opportunity for researchers to leverage the video medium to communicate their research...their contribution. It offers a 5-step process and several examples for creating effective research videos... botanist and oceanographer who has a blog and several videos on sharing your research through video. ...

...composed of actin and myosin which interact with each other to generate forces that cause cells and muscles...signalling pathway, which is a key regulator of all bilateral animal development. SHH plays a key role in proper...into the right and left hemisphere. Addgene has several plasmids that contain SHH, you can find them all...that are necessary for myosin activity and thus interaction with actin in nonmuscle systems in the fly. Spaghetti...other hand, is important in the formation of the visceral mesoderm which differentiates into the muscles...Embryos that lack bagpipe have severely disrupted visceral mesoderm and subsequently the gut does not form...is required for specification of the heart and visceral muscles in Drosophila." Development118.3 (1993...

...directly into at least one and preferably more than one of these five general purpose repositories. RSpace...own pros and cons. All ELNs have the virtue of liberating data from paper into an electronic environment...data out of the ELN; and (2) facilitate inter-operability with other research tools and infrastructure ...of Edinburgh, the University of Goettingen, and several other large research institutions. The following...repositories Over the past decade or so a number of general purpose repositories have been launched and are... associated with publications into one of five general purpose open source data repositories: Dataverse...one foot on the other side of the pond through interacting with colleagues in Edinburgh. You can follow ...

...lines in tissue cultures takes half a year, and generating a transgenic plant can take even longer, a transient...are investigating how the production of cancer therapeutic drugs, vinblastine and vincristine, are regulated...transgenic hairy roots takes at least six months. Generating a transgenic plant takes even longer, is more... that neither a stable cell line or plant is regenerated from the transformed tissue. Gene function is...by working with young seedlings and optimizing several other aspects of the transient transformation procedure...Once we were on the right track, we switched to luciferases to more easily quantify the effect of the different...GUS::tMAS to confirm transformation success. Luciferases are great quantitative reporters and use a substrate...

... are several factors that may prevent your gene from being up-regulated. For instance, generally, the ...known functions has the potential to be used in therapeutics, as it can increase gene expression at certain...be used to map molecular pathways and genetic interactions. Additionally, large scale CRISPR-based screens...Cas9 cutting activity, off target effects are generally not regarded as being a large problem for Cas9...driving aberrant transcription. That being said, we generally pick guides by putting the promoter of the gene...innovative technologies. To date his group has develop several popular CRISPR-based tools for altering DNA sequence..., Garnett MJ (2019) Prioritization of cancer therapeutic targets using CRISPR–Cas9 screens. Nature 568...

...them remain uncharacterized. This represents a considerable barrier to furthering research and developing...Cancer Center tackled this problem by developing a moderate throughput functional genomic platform to annotate...over 1,000 mutations, and the group has deposited several hundred plasmids from this study with Addgene. ...research team identified 1,049 perturbations in several genes spanning 33 cancer types. The types of mutations...indels (Ng et al., 2018). From this data, they generated lentiviral constructs using the High-Throughput...cell lines and any mutations that that confer proliferation advantages will be revealed in the absence of...is important for the development of targeted therapeutic interventions for cancer. This screening platform...

... gene expression in yeast Megan McClean’s lab generated an optogenetic toolkit that controls repression...frequency of genome-wide off-target DNA editing. Using several rapid and cost-effective screening methods, David...with reduced Cas9-independent deamination. They generated these editors using the BE4max architecture. The...reported in this study have ~10-100-fold lower average Cas9-independent off-target DNA editing, ~5–50-...off-target DNA editing, and maintain ~50–90% of average on-target DNA-editing levels relative to BE4max...division, and cellular differentiation, but it takes several weeks to delete or deplete individual genes. Michael...depending on transgene design and delivery. They generated hPSC lines with AAVS1-integrated, inducible, and...

... visualize their activity. In 2019, the first generation of norepinephrine sensors was developed, named...norepinephrine (Feng et al., 2019). But this first generation of tools had limited sensitivity and speed and... models of nLightG (left) and nLightR (right) generated using ColabFold (Mirdita et al., 2022). B, Representative...Speed One of the major advantages of this next generation of norepinephrine sensors is their speed. nLightG...GRABNE were more sensitive than nLightR, both generating a measurable signal after electrical stimulation...test point, ten pulses. This is in line with the general findings that red fluorescent proteins tend to ...improvements. In particular, they’re focused on generating variants with improved affinity and dynamic range...