...Virus Genome Interactions Important for Vector Production and Transduction. Human Gene Therapy 31:499–511...and Rep40) Rep proteins. Alternative splicing generates the different C-terminal regions of the Rep proteins...transfer plasmid, which contain regions of dsDNA. To generate a 3’ hydroxyl group needed for DNA replication...in the ITRs, uses its endonuclease activity to generates a ssDNA substrate which is then nicked by its ...splicing and differing translational start sites generate the three VP proteins. The three VP proteins assemble...Adeno-Associated Virus (AAV) as a Vector for Gene Therapy. BioDrugs 31:317–334 . https://doi.org/10.1007/...Improving the Efficacy of AAV Vectors. Molecular Therapy - Methods & Clinical Development 12:248–265 . https...

...genome engineering - such as certain forms of gene therapy - avoiding the use of viruses is also important...lab screened many SB tranposase variants for hyperactivity in mammalian cells. Their efforts resulted in...approximately 100-fold more efficient than the first-generation transposase (as determined by transposition assays...construct. This new tool kit allows the rapid generation of stable transgenic cell lines which can robustly... CD19-directed chimeric antigen receptor (CAR) therapy. SB is a valuable tool and an alternative to CRISPRs... Optimized Sleeping Beauty transposons rapidly generate stable transgenic cell lines. Biotechnol J. 2015...Mates L, et al. Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable...

...through the proliferation of specific, and not all, Langerhans cells, to create foci of proliferating cells....Brainbow The Lichtman group developed the original iteration of this technology in 2007. Used to label neurons... is expressed. Cre-mediated deletion of one or several of these genes permits the expression of genes ...occurs. Following the development of Brainbow 1.0, several more refined versions have been developed, which...tracing in zebrafish with Zebrabow Before the generation of Zebrabow (Pan et al., 2011), Brainbow was ...Tissue-specific Cre recombination in this mouse generates cells that randomly express either nuclear GFP...become a powerful tool for understanding cell proliferation. Inducible XFP expression in the Ubow mouse ...

...edited. For many experiments, the end goal is to generate a clonal cell line, so it’s important to verify... for a needle in a haystack before going on to generate and screen individual clones. If a clonal line...al., NAR). This technique uses the trace file generated in Sanger sequencing and a simple algorithm to...with your sgRNA sequence. The TIDE software will generate a graph representing all insertions and deletions... 4 clones. Did you use a dual guide system to generate your knockout? Then you’re in luck! You don’t ... to visualize an edit. Most dual guide systems generate a deletion large enough that it can be visualized...pair edit with a prime editor. What next? When generating a knock-in mutation, the best screening method...

...guide RNAs may tolerate a mismatch in the binding sequence, we recommend changing several bases as close... to as ‘indels’. While this is convenient for generating gene knockouts, NHEJ repair does not allow introduction...introduction of specific sequence changes. To generate a specific sequence change the cell must undergo...other groups have focused on strategies to improve overall HDR rates, we have shown recently that desired ...desired genome-editing events by HDR can be generated more efficiently by (1) improving editing accuracy by ...systematically characterize this phenomenon and found a general relationship between the distance of a mutation...probabilities our data revealed, and found that for generating a homozygous HDR event with optimal efficiency...

...which chews back the 5’ ends of the fragment, generating long overhangs which allows the single stranded...stranded regions with homology to anneal a polymerase, to fill in the gaps a DNA ligase, to seal the nicks...enzymes is that they can all work at the same temperature, so the entire reaction takes an hour or less...advantage is that this process makes it easy to generate wild type and mutant constructs at the same time...an overlap of 15-40 bp, with a primer melting temperature greater than 48℃. Both Snapgene and NEB have ...instead of using the exonuclease feature of T4 DNA polymerase, which must be controlled by the presence or ...methods that have been described--CPEC (circular polymerase extension cloning) and SLiCE (Seamless Ligation...

...important consideration is whether the affinity of an experimental protein-ligand interaction approaches...non-specific binding). Historically, glutathione-S-transferase (GST) fusion proteins have been immobilized on... target protein with the vitamin biotin. The interaction between biotin and the egg-white protein avidin...affinity (i.e. sub-nanomolar) protein-ligand interactions. Avidin and its derivatives have four biotin-binding...involves amine-reactive biotin derivatives that generate covalent bonds with positively-charged amino acids...solution was to exploit the best of both worlds. We generated a plasmid that produces biotin ligase and a protein...erythropoietin receptor and subsequently evaluated interactions with mutant and wild-type erythropoietin molecules...

...we believe it is best to further reduce onsite operations for the coming week. In an abundance of caution...Addgene charges enough for our services to run our operations. We don’t raise money except occasionally for...to launch a new service or product. Our daily operations for all ongoing processes are entirely self-supporting...they grasped the crucial role Addgene plays in accelerating research. Yet this was still not enough to ensure...funding organizations to request support for our operations until our normal work resumed. Addgene distributes...type of biomedical science research. Our work accelerates and enhances productive collaborations as well...distribution of plasmids for COVID-19 research is accelerating essential research in laboratories all around...

...Over the past several months, we’ve seen the emergence of several SARS-CoV-2 variants coincide with increased...the world” says Balazs. “By combining those with [sera] we got from people that have been given the vaccine...relative ability to neutralize.”   Figure 2: Sera used in the study came from individuals who received... two doses of the Pfizer or Modern vaccine. This sera was used in the high-throughput SARS-CoV-2 pseudovirus.... In their assay, the pseudoviruses express a luciferase reporter which is used as a readout for infection...Figure 3: Neutralization outcomes of convalescent sera with various pseudotyped virus. Image from Garcia-Beltran...quick to deposit their plasmids at Addgene to accelerate ongoing COVID-19 research. “I hope that by sharing...

...The contents of this post are intended to convey general information only and not to provide legal advice...you think about your career (and your future in general), keep in mind that you may have years to work ...for recent Ph.D. graduates). Therefore, it is imperative to plan for and develop your career into a series...Order which sets broad policy intentions directing federal agencies to propose reforms to the H-1B visa system...collaboration management, grant administration, clinical operations, and so on. Candidates should look broadly at...and your continued work is necessary to create therapeutics to cure the cancer. Authorship:  Publishing ...Interest of the EB-1A. Furthermore, the ability to leverage the prestige of the employer makes EB-1Bs a very...

...corresponding sequencing quality. Generally, you want to have 100x-1000x coverage of your library: in other words...transformation and amplification protocol, but the overall workflow is similar for most libraries. Here are...of the final recovered DNA. This bias can be counteracted by growing the cells on solid plates, where ... extraction. Prepare library product for next-generation sequencing Once the DNA has been extracted, the...of the library should be confirmed using next-generation sequencing (NGS). For gRNA libraries, you’ll ...using both pre- and post-amplification DNA and generates PCR sequencing products from both. These products...post has helped clarify some of the important considerations when using, amplifying, and verifying pooled...

... is the Arabidopsis thaliana - Pantoea agglomerans interaction. But the shape and metabolism of leaves...anticoagulant drug warfarin. In past research, several VKOR variants have been found that affect the body...postdoctoral work in Lin Tian’s lab at UC Davis is to generate a genetically encoded fluorescent sensor for serotonin...but these drugs are often ineffective or require several rounds of dose and drug adjustments. Unger ultimately...to study plant-bacteria and bacteria-bacteria interactions  Michal Bernach is a graduate student in Mitja...University of Canterbury where he studies how bacteria interact with each other and plant surfaces. His system...leaves can be changed by light and temperature and leaf autofluorescence can interfere with microscopy. Thus...

...Cas9 in an attempt to create gene-edited babies. Several hours later, the scientist, Jiankiu He, confirmed...such edited babies were born in a twin live birth several weeks previously. While this claim has yet to be...public consensus on genome editing, there was a generally agreed-upon international moratorium on utilizing...with healthy mitochondria, with the intent to eradicate mitochondrial diseases. The key success of the...issues raised around mitochondrial replacement therapies. In other words, rather than having a meeting...bedside is, in addition to thorough ethical considerations, in making sure that the research has been ...simulations, in vitro research, animal models, and several stages of human clinical trials. This model also...

... to have its genome fully sequenced There are several human disease models established in yeast, including... new at all, but it’s not as well-known in the general research community. Phillips Petroleum Company...release the K. phaffii expression system to the general research community in the 1990s when they abandoned...short, K. phaffii is a non-pathogenic yeast that operates as an obligate aerobe. It’s also a methylotropic...Thus, the production of glycoproteins for human therapies, such as antibodies, is possible in K. phaffii...peroxisome studies since their peroxisomes will proliferate upon a media change into methanol and peroxisomal...peroxisomal studies on biogenesis, degradation, and proliferation. Genome manipulation K. phaffii’s genome has...

...scientific community. Recently, the Zhang lab generated a TF library (aka the MORF Collection) and an ...isoforms transcribed from these sites. The Zhang lab generated a comprehensive library of each unique TF open... representing 92% of the MORF library, with an average of 206 cells per TF ORF. From this large sequencing...map of possible TF-induced cellular states was generated (Joung et al., 2023). Fig. 1: Barcoded ...then compared to that of reference cell types generated by the human fetal expression atlas. Computational... Good disease models can often be difficult to generate, in part because of difficulty with replicating...and a program that any researcher could use to generate the relevant disease model. Accurate modeling ...

...requested from Addgene psPAX2  - psPAX2 is a 2nd generation lentiviral packaging plasmid from the Didier ...and tet genes. It can be used with 2nd or 3rd generation lentiviral vectors and an envelope expressing...BB)-2A-GFP (PX458). lentiCRISPR v2 - This 3rd generation lentiviral transfer vector also from the Zhang...founding. Find pCMV-VSV-G. pRSV-Rev - This 3rd generation lentiviral packaging plasmid from the Trono lab...lab. Find pRSV-Rev. pMDLg/pRRE - This is a 3rd generation lentiviral packaging plasmid from the Trono lab...This plasmid from David Sabatini’s lab is a 3rd generation lentiviral vector that contains a scrambled shRNA... This vector from the David Root lab is a 3rd generation lentiviral transfer plasmid for cloning and expressing...

...enhanced site accessibility In order to generate a genomic alteration at a specific location, it must be accessible...appropriate for your specific research question. Considerations when choosing your Cas nuclease When choosing... off-target cleavage should be an important consideration during any experiment. Sensitivity Cas9 sensitivity...Comic by Maya Kostman. Another important consideration when choosing a Cas9 is balancing specificity...Jennifer Doudna Labs used targeted mutagenesis to generate hyper accurate Cas9 (HypaCas9). This nuclease ...the Jungjoon Lee Lab used directed evolution to generate Sniper-Cas9 resulting in a Cas9 with improved ...accessibility, specificity, and sensitivity are key considerations in choosing a Cas protein for your experiment...

...contributed by Jennifer Tsang AAVs are a great gene therapy delivery mechanism, but their small packaging capacity...splitting removes any exogenous sequences and regenerates a native peptide bond at the split site. ...tested the base editors in vivo and achieved therapeutically relevant editing efficiencies in the mouse ...comparable to those currently used in human gene therapy trials. They further tested the base editors by...Niemann-Pick disease type C, directly demonstrating the therapeutic potential for the split base editors. Find the...conversion of CO2 into water and electrical energy generation. Alistair McCormick’s lab recently developed ... Using parts from this kit, the McCormick lab generated knock-out and knock-in mutants, determined protein...

...assumption into question. Specifically, CRISPR-generated mutants believed to be knockouts can maintain ...Morgens et al., 2016). Only upon combining the data generated from both methods did a more comprehensive gene...Consequently, I will focus on different approaches and considerations present at the first stage of an RNAi-based... of the RNAi trigger requires a few initial considerations, such as the starting material (DNA-based vector...prevalent method due to ease of use and capacity to generate a long-term RNAi phenotype. Long hairpin RNA The...Importantly, instead of the assortment of siRNAs generated from a long hpRNA trigger, only a single ~21-nt...amiRNA). Although downstream work is required, several vectors can be found at Addgene that could serve...

...validated? This post will go over some of the general considerations you should review before using an antibody...important to know what you are aiming to achieve. In general, these are three core goals of antibody validation...different applications. Here, I want to highlight the general suggestions given by the International Working ...these pillars is that they are intentionally generalizable and not specific to certain applications or ...analysis of transcriptomic or proteomic data from several different tissues or cell types. If your antibody...the brain, but almost none in the liver (A). In general, RNA levels tend to correspond to protein levels...s important to understand if protein:protein interactions could be a factor in your analysis. Example:...

...enter postdoctoral training. Similarly, there are several factors to consider when selecting a postdoc mentor...system more, you will have a better idea of how to leverage it to ask new and interesting questions in your...confidence in the reagents and outcomes. This is preferable to finding out you've been doing something all...exploration, discovering new information. There are literally infinite questions one could pursue and experiments...the data and any recent relevant studies in the literature. Plus, presentations benefit from practice. If...better slides that more effectively tell a story? Generally, the answer to the second question is yes. As ...and how your research fits in with the current literature. Finally, if you give your talk or poster to ...

... 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....

...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...

...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...

... 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 ...

...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...

... 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...