...size, multimers may exhibit reduced transformation and transfection efficiency compared to the monomeric...1981). E. coli strains such as DH5α, HB101, NEB Stable, Stbl3, etc., are recA- mutant strains, and therefore... You can then gel extract the linearized band, ligate the plasmid, and re-transform the ligated plasmid... Elfinger M, Hartl D, Rudolph C. Transgene expression of transfected supercoiled plasmid DNA concatemers...multimeric forms (e.g. concatemers, dimers, trimers, tetramers). Since then, multimerization has been observed...the repeated plasmid sequence, giving greater concentration of cut DNA without changing the size. As a result...   Grow plasmids in recombinase-deficient strains Since plasmid multimerization is caused by homologous...

...RNA cargo into their targets makes them attractive and tractable tools for genome engineering. Scientists... Packaging Capacity (>8kb) High Transduction Efficiency Transient Gene Expression High Immunogenecity...alternatives? Well, for one they have a high transduction efficiency and can effectively infect a wide...of cell types that are typically ‘difficult to transfect’, such as primary cells. They can also be directly...organisms. Finally, viral delivery is typically straightforward across applications, while electroporation ...derived from viruses that infect people in nature, extra safety measures, like replication incompetent viral...through nuclear localization signals which can traffic their viral particles inside the nucleus, without...

...fluor. Transcribing and translating a protein fluorophore also takes time - bypassing translation altogether...expression strategies.” Chem Biol. 2015 May 21;22(5):649-60. PubMed PMID: 26000751. PubMed Central PMCID: ... PubMed Central PMCID: PMC3279232. 10. Song, Shiping, et al. “Aptamer-based biosensors.” TrAC Trends in...have used antibodies for diagnostics know how frustrating it can be to get a “bad batch”); Customizable...help reduce such background, thereby improving traditional visualization techniques. In addition, their ...biosensors. Aptabeacons Fluorescence resonance energy transfer (FRET) technology has been studied and used in...fluorescence-activated cell sorting (FACS) of E. coli transformed with aptamer libraries, the Jaffrey group selected...

...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 ...double-stranded DNA cleavage." Nature 533.7603 (2016): 420. PubMed PMID: 27096365. PubMed Central PMCID...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 ...TadA*) that satisfactorily accepts DNA as its substrate (see figure 2).          The artificially evolved...

...RNA polymerase III transcript, the sgRNAs were encoded on the lentiviral minus strand in the 3’ LTR. These... Figure 6: Illustration of split fluorophore assay to test reconstitution via mRNA trans-splicing. A ... with tRNAs, which are cleaved out of the RNA transcript by endogenous processing enzymes (Figure 1). ...including modifications to the Cas9 or reverse transcriptase enzymes, inhibition of host cell proteins, and...(Figure 3).      Figure 3: HeLa cells transduced with SARS-CoV-2 nucleocapsid protein (Plasmid...modification of microglial immune cells is critical for central nervous system research; however, microglia are...are notoriously resistant to viral transduction. Through in vivo screening of human promoters, the Zhang...

...reporter of transfection, which only report the efficiency of plasmid delivery to a cell. In contrast, TREE ... 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...At the same time Cas9n cleaves the opposing DNA strain which results in two DSBs that can be repaired ...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...

...examples I’m personally interested in include: intracranial self-stimulation (ICSS) and place preference... a behavioral experiment in which an animal is trained to “freeze” upon anticipation of a mild electric...(viral expression, efficiency of laser light transmission to the target neurons, appropriate stimulation...mistakes. The easiest way to learn optogenetics is straight through the source - find a lab within your own...optogenetics is performed in this species. However, transgenic rat technology is improving and may one day reach... reach the sophistication of mouse transgenics. Everything that has been described is intended for the...References 1. Stuber, Garret D., et al. "Excitatory transmission from the amygdala to nucleus accumbens facilitates...

...increases when target DNA strand separation is stabilized (2,3). Stable strand separation is maintained...and therefore destabilize basal strand separation. This decreased strand separation combined with the weak... Figure 1: Strand separation and target DNA binding to the Cas9 nuclease. Stable strand separation is...the non-target strand and the HNH/RuvC groove, and interactions between the target strand and the gRNA....interactions : 1. Interactions between the non-target strand and a positively charged groove formed by the Cas9...nuclease domains 2. Interactions between the target strand and the gRNA (see figure below) Because off-target...by a technique for detecting genome-wide double strand breaks (BLESS), revealed that these mutants do ...

... cell types. Specific transgene expression can be also achieved in nontransgenic animals by using cis ...population.  Combinatorial strategies can also be used for retrograde tracing: visualizing neurons that...delivery by increasing transgene cargo capacity, enhancing anterograde transport efficiency, and engineering...and Development workshop aimed at brainstorming strategies for using combinations of AAV-based molecular... cell of interest: AAV tropism and route of administration AAV tropism is defined by the viral capsid ...proteins that determine the AAV serotype. During transduction, the viral capsid proteins bind to the target...serotype used for a specific cell type. Find some transduction characteristics of select AAV serotypes in Table...

...STPF) at the Veterans Health Administration, I could never get one straight answer about what science policy...early demonstrates not only your interest to future positions, but will also make your transition from ...Technology Policy Fellow at the Veterans Health Administration. “But, seriously, what even is science policy...first few decades of my life following a very straightforward conveyor belt from high school to college to...professionals that entered the field from a more traditional policy background, learning the science part ... or LinkedIn to learn more about their career trajectory. If there’s one thing everyone loves, it’s talking...through convening activities, formal lobbying, or strategy setting.  The “little p” policy is non-governmental...

...enzyme or transcriptional activation, conformational changes, translation, and protein translocation. Targeting...be expressed. Are you looking at an intracellular or extracellular molecule? By fusing a biosensor to ...biosensor, you first need to decide the best way to track or measure the process you’re interested in. Depending... reaction, you could measure consumption of a substrate OR production of a product OR enzyme activity....biosensors Addgene has many sensors that measure the concentration of small molecules like arabinose or hydrogen...fluorescent protein fused to a domain that senses the concentration of a given ion - we’ll use calcium biosensors...triggers CaM/M13 interaction that increases FRET transfer between the fluorescent proteins. FRET is highly...

...would be transcribed and translated. For an N-terminally tagged protein, we used the same strategy, inserting... copy. The coding sequence for GFP is transcribed and translated along with the gene, producing both a...destroyed by CRISPR/Cas9 during transfection. Making the double strand break and introducing the tag through...Science Plasmid Collection Fluorescent tagging strategy Figure 1: Gene editing is the addition ... plasmid Figure 2: Donor plasmid design strategy. A donor plasmid contains 1 kb homology arms matching...base pairs on each side, in fact. Later, when we transfect the cells, these large regions of homology enable...directed repair (HDR) process following the double strand break CRISPR/Cas9 makes at the target site. The...

...any of these standard strains, choose other, indicate strain in the “Growth Strain Other” field, and contact...easy — our tech transfer team will communicate with your organization’s technology transfer office or other...room temperature. Growth Strain Please indicate which growth strain Addgene should propagate your...Survival strain. For plasmids that contain the R6Kγ origin of replication, choose the Pir1 strain. If your... you send your samples to Addgene pre-transformed in this strain. Hazardous If the plasmid will ... Send plasmid information to Addgene Material Transfer Agreement (MTA) approval  Send plasmids to Addgene...nourseothricin (clonNat), spectinomycin, streptomycin, tetracycline, combinations of the above, or other. High...

...certainly possible to select for desirable traits using traditional plant breeding approaches, these techniques... cells. In contrast to homology directed repair, these methods do not involve double strand break (DSB...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...

... less selective if used in intracellular labeling because the intracellular compartment may contain cross-reacting...using: different blocking reagents; titration of antibody concentration (this can also save money!); and ...method is transfection of short interfering RNA (siRNA). Knockdown caused by siRNA is also transient, and ...interest. This approach often involves the transient transfection of expression plasmids in a given cell ...for other sample types and for non-surface (intracellular) antigens. Unfortunately, antibodies frequently...commercially available. Various proteomic and transcriptomic datasets can be used to select a candidate ...This can involve cell surface labeling and/or intracellular labeling (or both). It is important to test ...

...activation of transcription in bacteria by co-opting σ54-based transcription. Transcription from σ54 promoters...enables researchers to maximize the chance of transducing a only a single gRNA per cell. The viral DNA ...genomic DNA. However, some cell types are poorly transduced by lentivirus, and thus screening with available...not possible. Primary murine T cells cannot be transduced by lentivirus and have been difficult to manipulate...Yusa Lab. This retroviral library was used to transduce T helper type 2 (Th2) cells from mice that constitutively...screening of cells that are refractory to lentiviral transduction, now enabling forward genetic screens in cells...cholesterol). However, size distribution and concentration of ApoB-LPs has been shown to have a strong ...

... deliver the substrate; however, secreted luciferase or the use of alternative substrate reagents can ...codon, which is then transfected into the desired cells. A hybrid mRNA transcript containing luciferase...luciferase, usually serves as an indicator of transcription within cells, where detection of the reporter...reaction requiring luciferin, ATP and oxygen as substrates. Production of photons by this bioluminescent...of Photon Generation Slower Faster Cofactors/Substrates Required Not Required Signal Strength Lower...Higher Lower Background Lower Higher Post-translational Modification Not Required Required Photobleaching...identified in various species with differing kinetics, substrate requirements and photon emission wavelengths. ...

...false positive colonies. Transformation - the linear fragments are transformed into E. coli and plasmid...common lab cloning strain has failed so far. What is most important is the transformation efficiency of the...assembly of a circular plasmid in vitro, before transforming it into E. coli for propagation. However, while...pathway that is present even in common lab cloning strains. This intrinsic bacterial recombination pathway...recombination reported in all laboratory E. coli strains tested so far. The use of this pathway as a cloning...1993). But why did this technique fail to gain traction? It is likely that both vector amplification with...using a simple two-step, 2 hr protocol prior to transformation (Figure 1). As this approach requires no special...

...al., 2025). In retrotransposon insertion, a combined endonuclease-reverse transcriptase uses homology ...fused to a shortened R2Tocc transposon protein, and can "retarget" the transposon to new loci in the genome...genome. The modified transposon RNA has untranslated regions (U, blue) on the outside of the homology arms...is one of the most important and abundant neurotransmitters, but it is challenging to monitor because ...rapidly taken up by postsynaptic receptors and transporters. The intensity-based Glutamate-Sensing Fluorescent... Image courtesy of IPI.   Repurposing retrotransposons for DNA editing By Emily P. Bentley For RNA-guided...beyond Cas9 with STITCHR: a combined CRISPR / retrotransposon system with the pithy name "site-specific target-primed...

..., the strategic use of whole phages as therapeutics, which is also gaining a lot of new traction lately...below). This initial transformation should be performed using a highly competent strain that you can use ..., scrape up, pool and extract plasmid DNA directly from the colonies. Transform the resultant plasmid ...homology-based searches return no candidates. Our strategy: Express random fragments of the phage genome ...enzymatic digestion, however, a physical fragmentation strategy is preferable. Nebulization involves forcing DNA...certain modifications to DNA bases.  Ligate and transform your resultant plasmid libraries into chemically...chemically competent E. coli and analyze transformants for appropriately sized inserts. We consider a library...