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...improvement comes with a trade-off: overextension past the reverse transcriptase (RT) template, resulting...presented oDNA: circular single-stranded DNA with short double-stranded regions just long enough for recombinase...metabolic comorbidities, demonstrating tunable, re-dosable control of therapeutic transgenes via endogenous ADAR...resolution PE6d: Processivity gains and overextension trade-offs The prime editor PE6d exhibits substantially... implicate specific residues in the reverse transcriptase polymerase domain as likely determinants of ...using a novel chemistry. This approach achieves ultra-low false-positive (0–9%) and false-negative (0–...types. 3. From bench to bedside: Therapeutic translation is accelerating Base editors and prime editors...

... recent works, transdifferentiation is usually carried out by expressing transcription factors specific...successfully transdifferentiated into neurons and β-cells [40-42]. Reprogramming and transdifferentiation: When...18818365. PubMed Central PMCID: PMC3987909. 6. Ban, H., et al., Efficient generation of transgene-free human...Cells. Stem Cells Transl Med, 2016. 5(5): p. 561-71. PubMed PMID: 27013738. PubMed Central PMCID: PMC4835250...PubMed Central PMCID: PMC4363833. 35. Eguizabal, C., et al., Dedifferentiation, transdifferentiation, and...PMID: 20364121. PubMed Central PMCID: PMC2877635. 39. Davis, R.L., H. Weintraub, and A.B. Lassar, Expression...redifferentiated to a different cell type. Transdifferentiation In this process, differentiated adult somatic...

...phosphatase-accessible. Kinase Translocation Reporters (KTRs) Kinase Translocation Reporters (KTRs) are genetically...particularly important for regulating signal transduction and measuring kinase activity at the single-...provides an alternative tool with their Kinase Translocation Reporters (KTRs) whose cellular localization...of two fluorescent proteins with very specific spectral properties that limit which fluorophores can be... A KTR is comprised of a fluorescently-tagged substrate for a kinase of interest which is further fused...activity is enhanced, leading to cytoplasmic translocation of the reporter. As a result, the KTR is shuttled... simple when fluorescent proteins with limited spectral overlap are used together. Second, KTRs are easy...

...solve the issue of “tiny vector, big transgene” by just straight up packaging an oversized genome of ~... successfully mediated transduction and expression of the full-length transgene in vitro and in vivo (...this, larger functional transgene products were produced following transduction.  So what’s going on here...overviews of three split vector strategies plus one additional split vector strategy for increasing the size ...between part A and B of the transgene to reconstitute the full-length transgene (Chamberlain et al., 2016...cells in mice. This vector transduced ~10% of cells, similar to the transduction rate of a single AAV. Odom...muscle physiological performance.  2. Trans-splicing Overview: For trans-plicing, splice site donor and acceptor...

...expressing a novel reverse transcriptase (RT), MarathonRT. Reverse transcriptases are critical tools for ...!  MarathonRT: A highly processive reverse transcriptase Article contributed by Will Arnold  Listen ...RTs in this regard and is even able to reverse transcribe the extremely long, highly structured HCV genome...structured template!). MarathonRT also faithfully transcribes RNA to DNA with low error rates compared to other... are dependent on long, error-free, reverse transcription of RNA such as long-read cDNA sequencing and...29109157. Nanobody toolkit to monitor retrograde transport at the cell surface Article contributed by Marcy...antibodies. These properties make nanobodies easier to transform and express in bacteria, which, in turn, makes...

... cross-neutralization occurs in the P.1 and B.1.351 variants tested. Image from Garcia-Beltran et al.,...high-throughput SARS-CoV-2 pseudovirus neutralization assay. Neutralization was quantified in terms of luminescence...Image from Garcia-Beltran et al., 2021 with permission.   Difference in neutralization after one dose...compare so many different strains. It was also the first to perform neutralization studies after just the...MIT, and Harvard, and his lab found that the neutralization effectiveness of current vaccines differed ...differed depending on the variants (Garcia-Beltran et al., 2021). Antibodies from individuals who received both... doses of the Moderna or Pfizer vaccine could neutralize many variants, including the B.1.1.7 (Alpha variant...

...restriction digest of plasmid DNA, and bacterial transformation. And more plasmid-related educational resources...reagent storage and inventory, managing orders, tracking students and research projects, electronic lab...share protcols, sign-up for shared equipment, and track inventory and orders. Quartzy caters to life science...features, and reference tables (i.e. antibiotic concentrations, commonly used primers). We also encourage ...

...surprising because in vivo transduction of AAV serotypes generally tracks between strains of mice. This suggests...PHP.B has a >40-fold increase in CNS transduction and transduces the majority of astrocytes and neurons... mice whose CNS was transduced by PHP.B but absent in mice who were not transduced by PHP.B. The Deverman...coding regions. In total, they tested 13 strains, with seven strains identified as permissive and six as non-permissive...enhanced PHP.B transduction rates. The group also determined the effect of mice strain specific Ly6a SNPs...permissiveness While PHP.B has impressive CNS transduction in some strains of mice, these results are not reproduced...in different mouse strains. The in vivo selection for PHP.B in C57BL/6J mouse strains likely contributed...

...Davidson and Roger Tsien Commemorative Travel Awards in 2017. These travel awards are intended to help early... Michael Davidson and Roger Tsien Commemorative Travel Awards: Melissa Chiasson, Elizabeth Unger, and ...mCherry ratios. Genomic DNA from each bin are then extracted and sequenced. Chiasson is currently using VAMP-seq... and in assays for measuring human serotonin transporter activity. Unger also plans to use the serotonin...constitutively fluorescent reporter can also be used to track how one population responds to a second population...

...research easier. I’m hoping my story demonstrates that transitioning from academia doesn’t have to mean ...better and make scientists’ lives easier. Why transition to Addgene? So why did I join Addgene? Was I ...weren’t some pretty hefty bumps during my graduate training. I particularly think that too many young scientists...do another thing I’ve always thoroughly enjoyed: travel. I’m actually finishing up this post on a plane...careers outside of academia during my graduate training. PhDs have many roles to play inside, on the outskirts...management, information distillation, etc) are transferable to many different fields. If you start thinking...

... focused ultrasound (FUS), intracorporeal (IC), intracerebroventricular (ICV), intramuscular (IM), intrathecal...Cre-dependent transgenes can also be paired with Cre-expressing transgenic mouse lines to restrict transgene expression...engineered to have enhanced function after intravenous administration, distributing widely throughout the organism...with broad CNS or PNS transduction, cell type specificity, or retrograde transport in neurons. (b) Genetic...capsids or transgenes. Sensory neurons and liver appear especially susceptible to off-target transgene expression-induced...differently in different mouse strains, a phenomenon that we are calling “strain-specific tropism.” For example...in many other strains, including FVB/NCrl, 129S1/SvImJ, DBA/2J, and C57BL/6J, the strain in which the ...

...controlled by titrating the transposase to transposon ratios. High ratios of transposase to transposon result...]. Like other transposons, piggyBac™ has two components, a transposon and a transposase. The piggyBac™...stable transgenic cell lines. The traditional method of making stable cell lines is to transfect, select... piggyBac™ transposon. These plasmids, when combined with a source of piggyBac™ transposase (available...distributor) allow you to quickly transfer a DNA sequence from the transposon vector to one of many TTAA sequences...folks at Transposagen. This post was contributed by Amar Singh and Mathew Goodwin from Transposagen What ...What is the piggyBac™ transposon? The piggyBac™ transposon was originally identified in moths in 1983, but...

...considered quite invasive. Restraint: anesthesia Location: trachea Gauge (tracheal tubing): 23–20 Volume: ...advanced Intranasal instillation Intranasal instillation is very similar to its intratracheal counterpart...Injection Route Restraint Location Gauge Volume Skill Intravenous Mechanical restrainer or anesthesia ...mL Expert Intratracheal Anesthesia Trachea 20–23 (tubing) < 0.07 mL Advanced Intranasal Anesthesia ...after initial mouse training. Other techniques require more skill and additional training, going from intermediate...biorender.com.   Needle techniques Intravenous Intravenous (IV) injections are most commonly administered...institutional oversight is required. Restraint: mechanical restraint or anesthesia Location: tail vein or...

...piggyBac (PB) transposon: The PB transposon is a mobile genetic element that transposes via a “cut and...Generates transgene-free and vector-free cells Limited genomic integration Multiple transfections required...Delivery DNA-Free High High Transgene-free and vector-free Multiple transfections required Protein Delivery...vector, but transient expression of Cre recombinase allows for deletion of the inserted transgenes that are.... This system is transfected into cells as a donor plasmid containing the transposon, along with a helper...sequences, which the PB transposase recognizes. Following reprogramming, transgenes can be removed by re-...promoters for transcriptional regulation, so it requires a different approach for regulating transgene expression...

...Ensure methanol concentration in transfer buffer is < 20% Wet transfer Increase transfer time Increase...large.  Transfer method   Wet transfers are more efficient, so if you’re struggling to transfer a large...consider using a wet transfer with a long transfer time. Be aware that the transfer works by electricity...decrease alcohol in transfer buffer Small proteins: Over-transferring Transfer with two membranes...increase alcohol in transfer buffer Mixed proteins: Transferring issues Transfer with two membranes...which increase as protein concentration increases, show the protein concentrations are within the antibody...commercially available options to choose from.  Transfer Transfers are a little trickier than gels and may require...

...and stable expression of the transgene   Transfer plasmids The transfer plasmid is the vector you clone...requires continuous administration of Dox to repress transgene expression. In contrast, in the Tet-on system...membrane pores) transfection. These techniques are still used and efficiently transfer plasmid DNA efficiently...comes to transferring DNA into primary cells or non-dividing cells. To target these un-transfectable cells...property makes the lentivirus an attractive choice for a gene transfer vector. Lentiviral vectors Although... purify and concentrate your lentiviral particles to get them to the optimum concentration to infect your...lentiviral transfer vectors The most widely used inducible lentiviral vector system is the tetracycline (Tet...

...contains the transposase gene (red) for expressing the transposase enzyme.2.) The transposase enzyme is ...which cuts the DNA.3.) The released transposon can now bind a strand of DNA with a TA dinucleotide (there...removal of the transposon; the plasmid is then degraded by the cell.4.) The transposase creates a double...double strand break in the DNA and allows the tranposon to integrate. Additionally, the TA sequence is duplicated....   The Sleeping Beauty transposon system The Sleeping Beauty (SB) transposon system is one such system...perform gene transfer in vertebrates. The system is comprised of 2 components: 1) an SB transposase, the enzyme...enzyme required for catalysis of transposition; and 2) a transposon containing the gene-expression cassette...

...experiment! Traveling from the Original Location - Axonal Transport It has been demonstrated that certain...cell type? Location (3): Traveling from the original location - Axonal Transport in the CNS What tissue...their efficiency at transducing different brain regions. Although AAV2 can transduce many tissue types,...axoplasmic transport. These AAVs may move toward the cell bodies of neurons (retrograde transport) or towards...retrograde and anterograde transport, but they also have similar transport characteristics (velocities...strong CNS tropism (4). If axonal transport is not desirable, transport can be reduced by injecting a smaller...scAAV) (7). As single stranded DNA viruses, AAVs normally require second strand synthesis before gene ...

... non-edited strand, they can cause the cell to remake that strand using the edited strand as the template...the nicked target strand. The initial result is a heteroduplex, with overlapping strands of edited and unedited...less than 10%. In contrast, the original paper introducing prime editing demonstrated ~20-50% efficiency...delivery strategy, cell type, and goal of the experiment can all impose different constraints and affect...Improving reverse transcriptase efficiency Building upon prior reverse transcriptase research, the Liu...the edit into one strand, there’s a mismatch between the original sequence on one strand and the edited ...other strand. To guide heteroduplex resolution to favor the edit, the Liu lab turned to a strategy they...