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...which ensures the editor will only be transiently present in transduced cells, limiting off-target editing... dCas9 fused to a DNA methyltransferase — that can drive lasting transcriptional silencing (Nuñez et al...virus-like particles (eVLPs) offers an option to transduce cells directly with ribonucleoprotein cargo. eVLPs...attB/P recombinase landing sites on opposite DNA strands. When the prime editor releases the DNA, the two.... (2024). Engineered virus-like particles for transient delivery of prime editor ribonucleoprotein complexes...Histone tail for Autoinhibition Release of Methyltransferase (CHARM), with several advantages over the ...  Rather than overexpressing an exogenous methyltransferase for epigenetic silencing, which can be toxic...

...variants (hereafter referred to as Cas9) and a single stranded guide RNA (gRNA) is sufficient to modify the genome...variants have been created in an effort to increase translation in a particular plant species or cell type (Belhaj...CRISPR components can be expressed stably or transiently depending on the delivery method and cell type...CRISPR components can be delivered and expressed transiently using a standard detergent, Polyethylene Glycol... More information on Agrobacterium-mediated transformation can be found in this blog post. The pDGE Dicot...Simplified schematic of agrobacterium-mediated transformation in plant cells. Agrobacterium tumefaciens can...plant genome. B) The Ti plasmid and helper are transformed into agrobacterium tumefaciens and exposed to...

...into smaller pieces, run on a gel, and transferred to a membrane for analysis through hybridization of ...researchers generating transgenic animals may choose Southern blotting to screen founder transgenic animals for ...techniques used to generate transgenic mouse lines, such as BAC transgenic or traditional knockins/knockouts... control sample. Southern blotting applications range from confirming the results of cloning or knockout...for the inserted transgene because Southern blots have a lower false positive rate than real-time PCR (4...hydroxyl group required for the extension of a DNA strand (8). Adding a small amount of a single radiolabelled...regular nucleotides results in the synthesis of DNA strands of all possible lengths because extension is terminated...

...proteins, which are directly trafficked to cell membranes. Membrane trafficking of farnesylated derivatives enables...Brainbow-3.0, but with mOrange2, EGFP and mKate2 as the fluorophores; mOrange2 is the default state. In... virus post-transcriptional regulatory element (WPRE), commonly used to increase transgene protein levels...fluorescent proteins: RFP (red), YFP (yellow) and M-CFP (membrane tethered cyan). Without Cre recombination, RFP...first fluorescent protein in the construct, OFP (orange), is expressed when Cre is absent; stochastic Cre...This system differs from Brainbow-1.0 in that transient Cre recombination is required to prevent constant... both fluorescence and sequence overlap (coral mOrange2, jellyfish EGFP, and sea anemone mKate2.) They...

...this FRET you speak of? Förster Resonance Energy Transfer (FRET) was originally described by Theodor Förster...alternative acronym Fluorescence Resonance Energy Transfer. Unlike the typical excitation and emission of...excited fluorophore, FRET involves a non-radiative transfer of energy (i.e. no emitted photons) from the excited...excitation by absorption of a photon 2. Energy transfer from the excited donor directly to the acceptor...the acceptor fluorophore. Otherwise the energy transferred from the donor will not be able to excite the...other and be oriented appropriately for energy transfer via dipole-dipole interaction. The efficiency ...biological processes occur within the typical FRET range, FRET efficiency is used to infer an interaction...

...concentration lies within the dynamic range of the curve. The dynamic range is the linear span between the lowest...the dynamic range of your standard curve, so if your sample does not fall within this range, it may be ... of the series dilutes by 1:2 for a series that ranges from 1:2–1:32. The specific dilution series used...Figure 1: A 1 mg/mL control is diluted in a series ranging from 1:2 to 1:32. Each individual step of the series... how well the data points fit the trendline. R2 ranges from 0 to 1 with 1 being a perfect fit and 0 indicating...that the curve can accurately measure.  Dynamic range depends on a number of factors, including the dilution...being used to measure the response. The dynamic range needs to be wide enough to cover all possible concentrations...

...members to the Board of Directors: Khandan Baradaran, Frank Stegmeier, and Luk Vandenberghe. We’re excited...members and get to know them! Khandan Baradaran, PhD Khandan Baradaran’s expertise in viral safety, gene therapy... with health authority regulations,” Baradaran says.  Baradaran’s path to science began in high school... getting these reagents is profound.” Frank Stegmeier, PhD Frank Stegmeier is the Chief Scientific Officer...in viral genetics. In regards to open science, Baradaran thinks the best way to solve problems is to work...think will get the research funded,” she says. Baradaran met Addgene Executive Director Joanne Kamens several...the targets that have the biggest potential to transform patients' lives. Previously Stegmeier was the ...

...process known as bioluminescent resonance energy transfer (BRET). The fluor then emits light at a tissue-penetrating... consists of NLuc fused to the more penetrative orange fluorescent protein fusion (OgNLuc). Both fluorescent...for excitation. The result was the cyan-excitable orange fluorescent protein, CyOFP1. CyOFP1 was molecularly...and improved maturation time compared to similar orange fluorescent proteins. In their work, the authors...great imaging tools generate cyan, yellow, green, orange and red fluorescence. The paper reports a wide ...Plasmid #85199), Yellow (YeNL; Plasmid #85201), Orange (OeNL; plasmid #85202) and Red (ReNL; plasmid #85203...have been made using fluorescent resonance energy transfer (FRET) probes. These probes generally consist ...

... affecting protein translation of different reporter proteins. Protein translation requires the start ...compared to CRISPR/Cas9 edits that rely on double strand DNA breaks. Due to this inefficiency it is crucial...constitutively expressed to identify successfully transfected cells. C-to-T editing by the APOBEC-Cas9 editosome...that results in the restoration of fluorescence. Transient reporter for editing enrichment (TREE) The Wang...Wang and Brafman lab developed a transient reporter for editing enrichment (TREE) to purify single base... to cells segregated solely by a reporter of transfection, which only report the efficiency of plasmid...frequency of base edits of interest than solely transfected cells. “Gene On” (GO)- a functional reporter ...

...72 hours post-transformation/transfection in a transient assay. For stable transformation, regenerated ...Agrobacterium-mediated transformation. We also constructed smaller vectors suitable for transient assays using ...changes a single base with an efficiency typically ranging from 25-75%, while the success of precise change...creates a nick in the DNA by cutting only a single strand, dramatically reducing the likelihood of detrimental...of the desired T-A base pair. Figure 1: Transition mutation (C>T, G>A, A>G, and T>C) installation...deaminases (ecTadA), fused it with dCas9, and transformed it into E. coli harboring the defective CamR ...using protoplast transformation. Cytosine base editors for plant There are several CBEs available for plant...

...attractive due to the ease of transduction in difficult-to-transfect cell types ranging from primary to embryonic... traditional transfection and random genomic integration of a plasmid. Viral transduction is also generally...integrated into the genome of the target cell or transiently expressed. It’s important to note that for all...vector can express your construct in target cells transiently or constitutively depending on the virus used...generally less toxic to cells than transfections, preserving cell viability. Library delivery Screening... entire genome, overexpression of all human transcription factors, barcoded libraries to trace cell lineage... with viral vectors instead of a traditional transfection method. This is because viral systems have a...

...Efficient gene transfer in C. elegans: extrachromosomal maintenance and integration of transforming sequences...easily manipulated with tools like CRISPR to make transgenic lines. The fruit fly (Drosophila melanogaster... in only 6 weeks compared to months to make a transgenic mouse line. Over the years the fly has become...DNA or RNA to edit their genomes or to create transgenic animals. Zebrafish are an ideal model organisms...development studies as their embryos are completely transparent. Thus scientists can easily observe early developmental...can be difficult in other vertebrate species. Transparency also allows scientists to easily observe fluorescently... control the temporal expression of different transgenes, the Stainer group determined that there are ...

...The development of the transient transfection platform for assaying transcriptional activity, for example...such as p300 and CBP histone acetyltransferases or CARM1 methyltransferase. This attendant signaling feature...dietary origin) that are diffused or actively transported from cells, traverse the body through the bloodstream...ligands NRs represent the largest family of transcription factors found in metazoans. The superfamily ... govern expression of genes involved in a broad range of reproductive, developmental, metabolic, and immune...multiple cell signaling systems. Activation of membrane receptors and signaling cascades conceivably allows...profiling datasets in to our data mining tool, Transcriptomine. We are currently nearing the completion of...

..., expressing Ad E1A and E1B, can be transfected with your transfer plasmid, carrying your gene of interest... performed 12–18 hours post-transfection to help remove toxic transfection reagents and keep the cells...helper plasmids, commonly called “triple plasmid transfection” (Figure 1).    Figure 1: Overview of key steps...cells happy. Harvesting virus Once you have transfected your cells, wait 2–5 days and then collect the supernatant...the concentration of viral particles that can transduce cells, which can vary based on target cells and... capsids, thus leading to an overestimate of transducible units in the virus preparation.  rAAVs  Now...Whitney, O., Walther, N., & Maurer, A. C. (2023). Transgene Expression in Cultured Cells Using Unpurified ...

...respondents (transfection method, amount of each component per transfection etc.). Take Pooran's NgAgo Survey...Doudna Lab Many thanks to our Guest Blogger Pooran Dewari! Pooran Dewari is a postdoc at the MRC Centre for...TOOL This post was contributed by guest blogger Pooran Dewari. Any views in this post are those of the...an ssDNA guide. Therefore, researchers must co-transfect cells with 5’-P-ssDNA guides and an NgAgo expression...delivery methods. Researchers can conveniently transfect/inject cells with in vitro preassembled Cas9/gRNA...). View the Complete and Up-to-Date Results of Pooran's NgAgo Survey The take-home message While most ...researchers will have to carry out repeated transfections with the ssDNA guide, although many may find...

...target and nontarget DNA strands upstream of the PAM site found in the nontarget strand. Protein Cas: ...sequences and keeping the array organized. Target strand: The strand of foreign DNA that Cas proteins recognize...recognize using an RNA guide. Non-target strand: The paired strand of foreign DNA cut by Cas9, whose sequence... and variable spacer sequences. The array is transcribed and processed into crRNAs, each including one...protospacer-adjacent motif” present in the non-target strand of DNA to be cut, which varies between CRISPR systems...from Addgene!   RNA crRNA: “CRISPR RNAs” are transcribed from the CRISPR array and include a spacer and...track down its suspect—the target DNA. tracrRNA: A “trans-activating CRISPR RNA” base pairs to the repeat ...

...between the edited strand and wild-type strand of the host DNA. When these strands hybridize, the nuclease...nuclease can detect strands with mismatches and cleave them. The results are then visualized using gel electrophoresis...is also key to perform these validations as translational researchers begin to use CRISPR as a therapy... search criteria are not exhaustive. Mismatch tolerance settings are often limited to off-target sites...nucleotides) that may still lead to off-target double-stranded breaks (Tsai et al. 2015). Additionally, current... genome-wide, unbiased identification of double-strand breaks (DSBs) evaluated by sequencing (GUIDE-seq...et al. 2013). GUIDE-seq and BLESS detect double-stranded breaks and do not require high sequencing read...

...different DNA strand comes along, it can attack this covalent bond thus joining the two DNA strands and releasing...planning to transform right away OR you can store the reaction at -20C overnight. 4. Transform TOPO Cloning...DNA sequence 5´-(C/T)CCTT-3' and digests double stranded DNA at this sequence. The energy from this breakage...stored as a covalent bond between the cleaved 3′ DNA strand and a tyrosyl residue of topoisomerase I (1). If...researcher to perform blue-white screening after transformation - self joining of the vector ends results in... minutes will not significantly improve the transformation efficiency).  5. Select and Analyze 10 White...add will depend on whether you are planning on transforming your reaction into chemically or electro-competent...

...scientists to quickly and easily generate zebrafish transgenic lines to study neurobiology and neuorodgenerative...pluripotent stem cells) through the expression of transcription factors OCT3/4, SOX2, KLF4, and c-MYC is the... method for generating iPSCs to express the transcription factors from a polycistronic cassette. However...comparable experimental results. Using PiggyBac transposons, the Woltjen Lab compared different variants ...reprogramming discrepancies. The polycistronic piggyBac transposon vectors tested in this work are available at ...approach Genetically modified organisms like transgenic mice are important tools for understanding the...a donor in FRET (fluorescence resonance energy transfer) and FLIM (Fluorescence Lifetime Imaging Microscopy...

...lentiviral vector to be transported retrograde and also has higher neuronal transduction efficiency and a better...virus to either restrict or broaden the host cell range (tropism). Pseudotyping is only done on enveloped...Lentiviral production uses three plasmids: (1) The transfer plasmid, (2) the envelope plasmid, and (3) the...the packaging plasmid. These are transfected into target cells which produce lentiviral particles.   ... titer is one of the reasons VSV-G is used to transduce stem cells and neurons. However, VSV-G’s broad...for off-target infectivity as it can bind and transduce many cell types before reaching the target cells...find tables and information on different viral membranes, tropism, and advantages and disadvantages: Table...