...sequences of DNA that can move around in a genome. In a laboratory setting, transposons can be used to both ...occurs which cuts the DNA.3.) The released transposon can now bind a strand of DNA with a TA dinucleotide (...transposon containing the gene-expression cassette that can translocate within the genome. The Sleeping Beauty...generation of stable transgenic cell lines which can robustly express your gene-of-interest, either constitutively...Additionally, in 2014 researchers at MD Anderson Cancer Center presented results from a clinical trial ...

...CRISPR components for plant genome engineering CRISPR can be used to knockout, activate or repress target genes...required. As with other model systems, multiple gRNAs can be expressed to modify several genomic loci at once...dCas9-VP64) or repressors (dCas9-KRAB or dCas9-SRDX) can also be used to activate or repress target genes ...result in a failed experiment. CRISPR components can be expressed stably or transiently depending on the...method and cell type in question. CRISPR components can be delivered and expressed transiently using a standard...information on Agrobacterium-mediated transformation can be found in this blog post. The pDGE Dicot Genome...transformation in plant cells. Agrobacterium tumefaciens can be used as a vehicle to deliver your gene of interest...

...editing and twin prime editing can integrate DNA cargoes of 80–100 bp, PASSIGE can integrate cargoes over a ...concentration and purity of the target molecule, which can be problematic for low-abundance targets or natively...plunge freezing By Rachel Leeson Cryo-EM samples can be damaged at the air-water interface (AWI) during...small proteins that naturally adsorb to the AWI, can protect cryo-EM samples from damage (Abe & Lim, 2024...air-water interface (AWI) before vitrification, they can be completely denaturated (left) or complexes may...of dCas9 fused to a DNA methyltransferase — that can drive lasting transcriptional silencing (Nuñez et...methyltransferase for epigenetic silencing, which can be toxic, CHARM uses a cofactor fused to a histone...

...mismatches that can nevertheless still lead to activity has been established, and can be used to identify...provide updates on how best to design gRNAs. You can also find validated gRNAs for your next experiment...Datatable. A more extended discussion of these subjects can be found in two recent review articles (Doench et...importance (target should be near the TSS but you can worry less about sequence optimality because you ...variants thereof offers additional PAM options that can greatly expand gRNA options. Created with ... in vitro transcription, or lentiviral delivery) can affect the accuracy of a predictive score, as well...computational prediction is ever perfect, but this can decrease the number of guides one needs to test in...

...binding factors (PUF). In a single step, researchers can now assemble designer PUF domains for RNA specificity...interesting because by changing certain amino acids you can change the specificity,” explained Zhanar Abil of...repressor. In principle, however, designer PUF proteins can be fused to other components to lend them many other...change the intracellular localization of RNA. “You can think of changing bases to other RNA bases or unnatural..., for example, by targeting pathways underlying cancer or other disease processes. Get the PUF Assembly...

...plants. In the most practical terms, what GreenGate can do is “save you half a year,” in comparison to more...fragments produced will have different overhangs, they can be designed such that they go back together only ... restriction endonuclease of choice, BsaI, which can be very sensitive to temperature fluctuations. He...sensitive protein,” he says. “There is nothing you can do about it but be gentle. If it no longer works,...

...within minutes of light exposure. Third, the response can be graded with light dose and is fully reversible...reversible after light retraction. Finally, because light can be applied non-invasively, its use is not restricted... endogenous cellular processes." The LITE system can now be applied to unraveling the genetic and epigenetic...time by making an AAV1 supernatant. (The details can be found here.) So tell us, readers, how will you... the brains of living mice both quickly and significantly. Genes under LITE control showed a rise in transcription...

...extra push, there’s evidence that laughter really can be good for you. A search on PubMed for the word ... few fun and very recent bits of evidence. (What can I say, we are scientists). Researchers in Taiwan ...those young people in a better mood. Laughter itself can sometimes even count as demanding exercise, and, ...Taiwan showed that a laughter program significantly decreased immunological markers of stress in adolescents...

...2) which can be utilized in three successive assembly steps. Before beginning, scientists can insert fragments...lab has engineered a modular mini-Tn5 vector that can be used to generate random mutagenesis libraries ...bacterium Pseudomonas putida and demonstrate that they can successfully create GFP fusion proteins with a variety... cytochrome c oxidase subunit VIII, so that they can be imported to the mitochondrial matrix. Gottlieb...pathway or induce other compensatory mechanisms. How can protein function be dissected while minimizing confounding... a membrane-permeable pharmacological agent that can rapidly induction heterodimerization of proteins ...the final construct. Up to eight entry vectors can be used for each Multisite Gateway compatible destination...

...monitoring localization; using FP timers, researchers can look at a single image of a cell and understand how...correlating this ratio to the total expression time can therefore be used to determine how long a pool of...for the theoretical timer shown in Figure 1B, you can use the equation fit to this linear region to solve... one benefit of the common FP timers is that you can monitor age by expressing a single FP-timer protein... oxygen, temperature, and pH, but timer activity can be calibrated to these different conditions, enabling...aggregation-prone tetrameric fluorescent proteins can be. Cytotoxicity, improper localization, and decreased...circuit (Saxena et al.) In a general sense, FP timers can be used in any situation where one wants to understand...

...more about these two new AAV capsids and how they can be useful tools for studying the CNS and PNS! What...expressing Cre in a cell type of interest, a scientist can isolate DNA from the infected animal and use the ...this barrier - only those that cross the barrier can be identified by PCR later. The PNS presents a different...AAV-PHP.S versus their parental AAV vectors. What can these AAV’s be used for?  1. Multi-color labeling...transactivator) is co-injected. The dose of this AAV can be adjusted to achieve sparse levels of labeling,...supplemental figure 8 of Chan et al). These new AAVs can serve as tools for basic science research (neuronal...multi-color labeling of neurons (figure 4). These capsids can serve as powerful cell-type-restricted gene vectors...

...efficiently investigate gene function. CRISPR libraries can be used to knock out, inhibit or activate target ...function while nuclease-deactivated Cas9 (dCas9) can be tethered to a repressor (CRISPRi) or an activation... are subject to an increase in dsDNA breaks that can cause cell death. Thus, CRISPR inhibition (CRISPRi...In addition to inhibiting gene expression, dCas9 can also be used for transcriptional activation. Activation...genome-wide gain of function screens. These screens can help reveal the function of lowly expressed genes...Tanenbaum et al., 2014). The sgRNA or tracrRNA sequences can also be modified to include RNA domains that recruit... screen, implying that ORF and CRISPRa libraries can be used in complement to study gene function. Find...

...sort of virulence as well — namely that they can cause cancer via insertional mutagenesis (which HIV does...simple spokesmodel for the whole family; however, you can use Figure 1 to compare the genomes of these two ...particle — and of the normal, replicative life cycle — can help you understand these seemingly odd choices and... of its first steps! However, retroviral vectors can still direct the translation of proteins because ...they have two dimerized linear ssRNA strands (which can have a different genomic sequence from one another...et al., 1971) and the full wild-type viral genome can direct the production, packaging, and release of ...viral particle, and how we as dedicated researchers can exploit it. This blog was written by Addgenie Brian...

...prime editing, whereby a multitude of configurations can be used to introduce specific target modifications...observed from high-throughput experiments, where we can evaluate tens of thousands of designs in parallel...the best pegRNA design for a specific edit, users can first define their edit in brackets and add the upstream...sequences to their input string (Figure 2). This string can then be used as input for the prediction on the website...), a model that predicts how well a genomic site can be edited based on its chromatin environment. After... pegRNA design with PRIDICT/PRIDICT2.0, ePRIDICT can help you understand how chromatin might influence...editing prediction models (such as PRIDICT/PRIDICT2.0) can greatly facilitate prime editing experiments, thus...

...Bimolecular Fluorescence Complementation (BiFC). It can provide a qualitative or quantitative readout of ...of the interaction in vivo or in vitro, and can be used for measuring protein expression or localization... When Protein A and B interact, the FP fragments can assemble the full structure of the fluorescent protein...from a different base FP (for example, EBFP2(1-10) can be used with GFP(11)). Some of the articles linked...color labeling with exogenous fluorophores, which can even be bright enough for single-molecule tracking...interest. Remember that the FP fragments themselves can sometimes interact in cells and produce background...termini are important for their interaction. You can read our previous blog post for more on BiFC principles...

...investigator needs a standard against which the results can be compared; results from an experiment lacking the...on to learn more about the various controls that can be used for plasmid-based experiments. What are control...expression level of Gene X in the untreated cells—but can we be certain? Do we know whether the plasmid delivered...the baseline/standard against which other samples can be compared. The Empty Vector Control (mentioned ...and biological. In general, technical replicates can be thought of as "plate controls". They are NOT independent...Conversely, biological replicates ARE independent and can be thought of as "reproducibility controls". Biological...successfully transfected our cells (Figure 3), we can continue with our experiment, incorporating additional...

...RNAs. To gain this specificity, the eukaryotic RNAP can recognize and bind to specific promoter elements....cytomegalovirus Constitutive  May contain an enhancer region. Can be silenced in some cell types. EF1a General expression...and several tetracycline operators. Transcription can be turned on or off depending on what tet transactivator...Inducible with galactose; repressible with glucose  Can be used independently or together. Regulated by GAL4...polymerase, which is also controlled by lac operator. Can be induced by IPTG. Commonly found in pET vectors...in the absense of lac repressor (lacI or lacIq). Can be induced by IPTG or lactose. Leaky promoter with...gene expression  Promoter from bacteriophage lambda Can be temperature regulatable  Often paired with the...

... they can scan for any signs of danger. In this post we’ll focus on how fluorescent proteins can and have...incremental change of the excitation wavelength can significantly affect emission intensities from fluorescent...coming from this volume. The consequence is that you can detect signal in thick specimens. See the video below...lymph node after infection with S. aureus (red). You can then see neutrophils swarming around S. aureus and...interactions. Even if virtually all fluorescent proteins can be used in your experiments, experience has shown...excitation wavelength (Salomonnson et al. 2012). They can also be excited efficiently at wavelengths between...the the Cre/lox recombination system. A researcher can flank fluorescent proteins in a plasmid with loxP...

...mismatches. However, some ligases can ligate mismatches to a significant degree, and very active ligases...dependent DNA ligase from a thermostable bacterium that can survive high temperatures (up to 95 °C) and is active...right ligase, conditions and suitable probes, LDR can reliably detect single-base differences. The original...visualized the ligation product in a gel, but we can now use fluorophore- quencher pairs or qPCR-based...ligases, such as T4 DNA Ligase, can ligate nicks containing one or more mismatches near the ligation junction...This “peeling back” of the 5 ́-phosphorylated base can be observed in the crystal structures of several ...26). Despite this higher fidelity, Taq DNA Ligase can still detectably ligate many T:G, T:T, and A:C mismatches...

...buy-in from all of Addgene’s teams. This solution can easily be implemented in an academic lab setting,...ambiguous terms. Defining words and applying annotation can be particularly useful in academic labs when creating...Clarify: Asking for clarification during a meeting can help get everyone on the same page. Others in the...meeting might also have the same question and asking can help the team identify possible points of confusion... done. But if you implement it well, here’s what can happen: More effective meetings. When everyone at...people aren’t listening to you or understanding you can be incredibly frustrating. Clarifying the words —...