...enhanced the ability of scientists to make genomic alterations, bringing about a revolution in genome engineering...engineering applications of CRISPR including: generating single or multi-gene knockouts in a wide variety...editing using homology directed repair (HDR), and generating large genomic deletions. What are the advantages...and specificity is a concern, such as knockout generation or homologous recombination.  However, experiments...step in this type of CRISPR experiment is the generation of the RNP complex.  While one option is to purchase...in vitro transcribed from ssDNA, which can be generated by commercial vendors such as IDT. These two components...electroporation (A in the figure above), which generates pores in the cell membrane, allowing for entry...

...neurons throughout the whole brain. Neurons of the peripheral nervous system (PNS), which connect the heart...mutant AAV capsid (cap) expressing plasmids is generated. Diversity is introduced into the cap plasmid ...was neurons and astrocytes. Why screen for IV deliverable AAVs? Typically AAVs are directly injected into...transduction efficiency of neurons were measured. On average, AAV.PHP.eB and AAV.PHP.S had 1.5- to 2-fold higher...They also transduced a higher number of neurons overall (GFP+ cells that also stained for a neuronal marker...labeling of neurons, because it doesn’t require the generation of a transgenic mouse. 2. Cell-type-specific...translational applications (mechanistic studies and gene therapy treatments for neurological diseases). The Gradinaru...

...technology and its advantages? Fujii: In the last several years, my lab has been working on development of...specific genomic regions while retaining molecular interactions. For this purpose, we developed the insertional... regions. I believe that enChIP will markedly accelerate research of regulation mechanisms of genome functions...some of its potential applications? Fujii: We generated a series of retroviral vectors expressing tagged...biological systems. We are currently developing several additional systems of enChIP to make the technology...to journals. In addition, it is my pleasure to interact with my colleagues in meetings of scientific societies... Rhine River. In the US, I very much enjoyed interactions with many excellent colleagues at NYU and visiting...

...implantation and generation of transgenic mice. Purification: GFP can be used as a general epitope tag for...biosensor or cell marker, studying protein-protein interactions, visualizing promoter activity, and much more...fluorescent activity; very little truncation is tolerated, however, point mutations are acceptable. GFP'...mutations have since been introduced to GFP and new iterations of fluorophores are constantly being engineered...energy transfer (FRET): This is used to study the interactions between two proteins or between two domains ... has also been used to study protein-protein interactions. In this case, two portions of EGFP are fused... their activity and visualize the effects of alterations to this activity. Learn more about optogenetics...

..., the legacy of each government and generation is in the veracity of its work. Alina Chan is a postdoctoral...these mistakes quickly. However, many scientists operate in a hyper-competitive environment where getting...speak out in unprecedented force during the 2015 federal election.   Even as we marched in solidarity with... Washington, DC, USA 38.9072°N 77.0369°W – Mark Lucera I am a passionate scientist.  I care about my ...People asked, and we talked.  That's a win. Mark Lucera is a PhD trained scientist at the University of...Postdoctoral Association.  Follow him on twitter at @Mark_Lucera. Ann Arbor, MI, USA 42.2808°N 83.7430°W – Alexandra...Aurora, CO where she studies the development and regeneration of mammalian taste buds. Erin is also the President...

...pEcCas/pEcgRNA is superior in several ways: The pEcCas plasmid can be operated at 37 ℃ for faster growth ...plasmid that expresses the S. pyogenes Cas9, a temperature-sensitive replicon for self-curing, an arabinose..., 2) changed the replicon of pCas into a non-temperature-sensitive replicon, and 3) added the sacB gene...containing BsaI restriction enzyme sites at its ends to generate pEcgRNA. The ccdB serves as a counterselection...genes of interest in BL21(DE3) using the newly generated pEcCas/pEcgRNA system.   Advantages of the pEcCas...directly ligated with the annealed 24 nt oligos to generate new pEcgRNA. pEcCas/pEcgRNA can be applied not... the gene dosage of glucose dehydrogenase and generated a strain that could achieve glucose dehydrogenase...

...more stable than the methyltransferase. When a plasmid is lost, the methyltransferase is degraded and/or...stabilized luciferase-expressing plasmids to examine in vivo-induced promoters in the Vibrio cholerae vaccine...ensure that plasmids are passed down through generations (Figure 1). These so called “plasmid addiction...composed of restriction endonucleases and methyltransferases. Restriction endonucleases cleave DNA at ...specific nucleotide sequences. To counter this, methyltransferases add methyl groups to bases at specific nucleotide...stably express antigens in sufficient quantities to generate an immune response. As a proof of concept, they...genes that couple host cell division to plasmid proliferation." Proceedings of the National Academy of Sciences...

...preps and our lentiviral preps.   Viruses are generated using standard methods that have been optimized...optimized for each specific virus in order to generate high quality preparations. After production, all virus...the University of Pennsylvania Vector Core. In general, titering is performed by the facility that produced...suggests that titers obtained using ddPCR are generally two to three fold higher than those achieved using... us. Full sequencing of the Viral Genome Next-generation sequencing is performed on viral genomes isolated...items) are produced in 293T cells using the 2nd generation psPAX2 and pMD2.G packaging system developed ...lentiviral preparations (“-LVC” catalog items) are generated from lentiviral preparations (described above)...

... developed, though, researchers at HuidaGene Therapeutics guessed that fusing MPG to an ABE would allow...transversion editor AYBEv3. This tool still retained considerable A → G editing activity at most sites, in addition...they co-expressed Polη, a translesion synthesis polymerase known to preferentially incorporate A bases across... N108Q mutation in the deaminase), produced an average A → C product purity of 48%. Like CGBEs, these ...abasic site before filling the empty space. Over several rounds of rational mutagenesis, the team progressed...allow it to recognize thymine (T) or cytosine (C). Several different teams succeeded with this approach around...recognize T and/or C substrates. The HuidaGene Therapeutics team has deposited their editors, called gTBE...

...processes from development to metabolism to fertility. Several zebrafish-specific gene expression databases that...available, new hypotheses about specific genetic interactions arise and tools to deactivate specific genes...way to deactivate genes without spending months generating mutants. Unexpectedly, phenotypes sometimes differ...of gene regulation (El-Brolosy et al., 2019). Generating zebrafish transgenics Creating transgenic organisms...different experimental conditions. Strategies for generating transgenic zebrafish are well established (Felker...cell RNA sequencing approaches (Harland, 2018). Several different methods have been developed, relying ...i.e., redundant) genome, their long three-month generation time and labor-intensive maintenance compared...

...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...

... replicates how you’d interact with team members in person, and improves overall communication.  Maintain...project and accelerates the pace at which problems can be addressed and new ideas generated. Furthermore...team in Paris, France. While efforts like these accelerate the progress of research, they can also present...different tasks. But these challenges can be exaggerated when communicating across time zones, digitally...the times that work best for them. This also accelerates the pace of research by circumventing the slower...exchange project at the International Cancer Immunotherapy Conference in Frankfurt, Germany When funding...

...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 ...

...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...

...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...

...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...

...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...

...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...

...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...