...breakage of both DNA strands - a double-strand break (DSB). DSBs can be caused by intracellular factors such ...CRISPR/Cas-mediated Transgenesis (2018). Pubmed. The Road Less Traveled: Strategies to Enhance the Frequency...overhang. This will serve as both a substrate for proteins required for strand invasion and a primer for DNA...DNA repair synthesis. The ssDNA strand then displaces one strand of the homologous DNA donor and can template...pathways: classical double-strand break repair (DSBR), synthesis-dependent strand-annealing (SDSA), and break-induced...dHJs are four-stranded branched structures that form when elongation of the invasive strand “captures” and...on the crossing strand (horizontally at the purple arrows) or on the non-crossing strand (vertically at...

...created the tetracycline-controlled transactivator (tTA). They fused TetR with the transcriptional activation... polymerase and transcription factors. Since the promoter region drives transcription of a target gene...repressor then prevents transcription from occurring, which means that transcription is now OFF.   We’ll...its native context, the tetracycline repressor (TetR) can bind to the tetracycline operator sequences (TetO...(TetO), preventing transcription. In the presence of tetracycline (Tet), TetR preferentially binds Tet...Response Element (TRE)), allowing transcription to proceed. When tetracycline or one of its derivatives is ...other. For example, the GAL4 transcription factor cannot drive transcription from the QUAS promoter, and...

...initiation step of gene transcription; today we'll provide an overview on how transcription stops, or termination...from the transcription machinery. Terminators are found downstream of the gene to be transcribed, and typically...generally serves to promote transcript longevity in eukaryotes and promote transcript degradation in prokaryotes...GC-rich hairpin in the RNA transcript followed by a weakly bound poly-uracil tract as shown in the figure ...thought to destabilize the transcription complex, initiating cleavage of the transcript.   Figure 1: ...Hepatitis Virus Posttranscriptional Regulatory Element Enhances Expression of Transgenes  Delivered by ...terminators in the context of AAV transgene expression levels. Transcription Termination Rho-dependent termination...

...gene transcription (6). The EBOV genome contains seven viral genes, each flanked by a transcription start...replicated and transcribed in the cell when the appropriate viral proteins are provided in trans (see Other...genes and the minigenome are transcribed (and the viral mRNAs are translated) by cellular machinery. NP ...altering the amount transfected (see Technical Tips). Minigenome replication and transcription In order to replicate...it will terminate transcription and polyadenylate the mRNA. These mRNAs are translated by cellular host... lines that are highly transfectable.  The amounts of each plasmid transfected into cells are also extremely...plasmid quality and transfection efficiency may vary between labs, initial titration experiments might ...

...recombineering substrates to avoid errors introduced during PCR. ssDNA Substrate ssDNA substrates can be generated...differ from traditional restriction enzyme cloning will be explained in greater detail. Substrate DNA design...   Substrate DNA Design and Generation Whether you use a linear dsDNA or ssDNA substrate depends ...ssDNA substrate is best for point mutations or changes of only a few base pairs.  dsDNA Substrate dsDNA...find out empirically which is the lagging strand. ssDNA substrate is more efficient than dsDNA with a recombination...approaches.    Bacterial strain with integrated defective prophage A number of E. coli strains exist that stably...red phage. One such strain is DY380, which is derived from the DH10B E. coli strain. Several other bacterial...

...process called transfection. Transfection is somewhat comparable to bacterial transformation (the introduction...the transfected plasmid to be expressed transiently. Since the DNA introduced in the transfection process...not guarantee 100% transfection efficiency. Stable transfection A stable transfection is used to create...reagents vary. Plasmid transfection into mammalian cells is fairly straightforward and the resultant cells...the genome to form a stable transfection. Unlike bacterial transformation, scientsts do not "select" for...fluorophore in a later post, so stay tuned! Transient transfection and the elusive "mammalian ORI" For many...plasmid depleted, but still want to use transient transfection methods. Since there are no "natural" mammalian...

...changing transfection ratios can affect transfection efficiency. For some transgenes, transfection optimization...containing structural genes and a transfer plasmid carrying the transfer gene. By dividing the viral elements... structural elements, envelope, and transfer gene are transfected into a packaging cell line, usually ...enough concentration, or titer, for your experiment. To improve titer, first optimize transfection conditions... lentivirus can be concentrated by a variety of methods such as ultracentrifugation, centrifugation on...volume than with the unconcentrated prep. For example, at Addgene we typically concentrate 100-fold meaning...preparations without ultracentrifugation. Sci Rep. 2015; 5:13875. PubMed PMID: 26348152. PubMed Central PMCID: PMC4562269...

...binding to the template strand (strand used by RNA polymerase II for transcription) are more effective for...days. The crRNA and tracrRNA are annealed on a PCR block and the annealed cr:tracrRNA is then mixed with...complex. This complete transfection mix, i.e. csRNP plus ssODN, is then transfected into stem cells. Cells...experiments. Cas9 protein, annealed cr:tracrRNA, and single-stranded donor DNA. The success of the epitope... efficiency, we have recently demonstrated that the two-part cr:tracrRNA system is much more potent than.... Not only is the cr:tracrRNA approach highly efficient, it is also straightforward as the RNA molecules...prepared with an in vitro-transcribed (IVT) sgRNA versus synthetic cr:tracrRNA. Cells that had successfully...

... complicated transition from a clump of cells to a fully differentiated body. In contrast, these dynamic...two-photon intravital microscopy from PLoS Pathogens - Kamenyeva et al, 2015. Cellular trafficking studies... skin. Two-photon intravital microscopy. The video above depicts the infiltration of neutrophils expressing...available for tracking certain cell types. Another useful tool for marking and tracking cells is the the... 18261937. PubMed Central PMCID: PMC2390753. 4. Egen, Jackson G., et al. "Intravital imaging reveals limited...PubMed Central PMCID: PMC4772972. 11. Salomonnson, Emma, et al. "Cell-based and in vivo spectral analysis...fluorescently labelled antibodies made it possible to track cells from one organ to another. Thus several labs...

...that increase transduction efficiency, none allow for simple and efficient transduction of both the CNS...capsids were packed with a GFP transgene and GFP intensity and transduction efficiency of neurons were measured...intensity compared to the strains they were derived from. They also transduced a higher number of neurons... Table 1: Transduction efficiency of AAV-PHP.B versus AAV-PHP.eB in the CNS. A GFP transgene was packaged... Table 2: Transduction efficiency of AAV.9 versus AAV.PHP.S in the PNS. A GFP transgene was packaged...the generation of a transgenic mouse. 2. Cell-type-specific restricted transgene expression in the CNS...introduce a transgene into a specific cell type in the brain, you needed to make a transgenic mouse. Combining...

...and synthetic biology have transformed the way scientists create new traits in bacteria and enabled the...addition to nucleotide-based linking strategies, post translational protein fusion would be advantageous...optimization of industrial yeast strains. However, industrial yeast strains are more difficult to genetically...be difficult to transform. To increase the genetic toolbox for industrial yeast strains, the Borodina Lab...lab from the University of Utah developed SapTrap. The SapTrap system uses golden gate assembly to produce...alone. Molecular processes from signal transduction to transport rely on protein-protein interactions and...be taken with common S288c strains since failing to fully complement strains with multiple auxotrophies...

...pEcgRNA system in 4 E. coli K-12 strains, 2 E. coli B strains, 1 E. coli W strain, and 1 Tatumella citrea, with...in E. coli BL21(DE3) as transformants failed to grow after the transformation of pTargetF into the BL21...applied not only in coli K-12 strains, but also in E. coli B and W strains. The optimized plasmid curing...pTnsABC expresses the transposases TnsA, TnsB, and TnsC pQCascade expresses the transposase TniQ, the Cas proteins...stable and high-yielding strain which had 6 times the yield of the industrial strain. Not only is MUCICAT ...pQCascade) with 4 rounds of transformation to complete the N-acetylglucosamine strain library construction in...multicopy integration. (B) Anhydrotetracycline (aTc) induces expression transposases leading to increased integration...

...a protein expression strain, we suggest retransforming the plasmid into a strain meant for DNA purification...incubate Check the bacterial strain Differences between E. coli host strains can impact plasmid yield. Check...to include an extra wash step or use a different strain. For most applications, Addgene uses and recommends...Stable to transform and prep high quality DNA that contains these elements. Be aware of endA+ strains Strains...incomplete. Protein expression strains are not good for cloning Bacterial strains adapted for protein purification...sounds like extra work, but it will likely save time in the long run! While the choice of strain will ultimately...antibiotic concentrations will work just fine; however, for low copy plasmids or slower growing strains, a change...

...reason, we speak of excitation and emission spectra. Both spectra possess clear maxima where excitation and...Fluorescence Resonance Energy Transfer (FRET).   Figure 1: Emission spectra and respective colors of...Excitation and emission spectra for APC and APC-Cy7. While the excitation spectra of both fluorophores show...Excitation and emission spectra for eGFP and FITC. As these fluorophores' spectra virtually overlap, you...cells in vitro. I transduced the cultured cells with a virus that encodes a transgenic protein for the ...having a separately expressed transduction reporter is that not every transduced cell will necessarily manage...the transgenic protein. We don't know yet how abundantly CD45 will be expressed on the transduced cells...

... by the bottom strand versus nicking the bottom strand first followed by the top strand. Both produced...increased concentration of Taq polymerase, 2) increased PCR extension time, 3) increased concentration of MgCl2...complementary mutant strand (step 2 and 3). Then the uracil-labeled template strand is degraded by uracil...present on both strands of the plasmid. The ssDNA template is generated by transforming a backbone phagemid...ExoI then fully degrades the nicked strand. Synthesize mutant strand: A mix of degenerate primers and the...Degrade wild-type template strand: Next, the opposite wild-type DNA strand is nicked by the BbvCI variant...step 1. Then this strand is degraded with ExoIII and ExoI. Synthesize 2nd mutant strand: The second mutant...

...from their compartment of action via sequestration. The strategy relies on several observations: Rapamycin...appear to mind extraneous proteins bound to its outer mitochondrial membrane. Sequestration is accomplished...GATEway Cloning Frustrated by the complexity of assembling recombination of transgenesis constructs? A ...the intracellular kinase domains into contact and initiating signaling. Grusch et al. demonstrate that...CRUSH vector in transgenic mice, the lab established a dual-color RNAi “sensor” mouse strain in which Cre...constructs constrained only by the ability of E. coli to maintain the final plasmid after transformation. Addgene...each transgene expresses one of three possible fluorescent proteins, chosen for minimal spectral overlap...

...vectors for selective transgene expression. While this is a powerful experimental strategy, one of the most...loxp pair, and flips the transgene into the correct orientation for transcription. Upon recombination one...end of the transgene. Since loxp and lox2272 can not recombine, this ensures that the transgene is no longer...Vectors). While this strategy greatly reduced the overall level of off-target transgene expression, low levels...flank a transgene (homologous pairs) or used different scrambled sequences to flank the transgenes (non-...Rumpel S (2013) Analysis of Transduction Efficiency, Tropism and Axonal Transport of AAV Serotypes 1, 2, 5...2020) Monosynaptic Tracing Success Depends Critically on Helper Virus Concentrations. Front Synaptic Neurosci...

...project forward, transductions should not be rushed into. Before you start any transduction, you should always...Primers targeting the transgene are also beneficial as they ensure that the correct transfer plasmid was used...qPCR based methods for viral titration consider using a reverse transcriptase mutant in a separate control...deficient in reverse transcriptase will produce low levels of virus and can be used to track carryover. Functional... positive for vector encoded transgene expression following transduction with serial dilutions of a viral...resistance genes carried on transfer plasmids. Target cells are transduced with serial dilutions of a ...can also impact transgene expression. The most accurate method of lentiviral titration uses qPCR to measure...

...the usual transfection drawbacks: transient expression and needing to work with a transfectable cell line...Cassettes transfection gRNAs expressed from a polycistronic transcript  Requires co-transfection of Cas9...Cassettes transfection gRNAs expressed from a polycistronic transcript Requires co-transfection of Cas9  ...expression platforms: transfection, lentiviral transduction, and AAV transduction.  In most of Zetsche ... rely on transfection of more than one vector to express the gRNAs and Cas9. Co-transfections can lead... GFP tag were transfected on a single plasmid. This is versus 2.4% of cells transfected with a pool of...Lentiviruses carry a (+) strand RNA copy of the DNA sequence, which is a suitable substrate for Cpf1. This means...

...Reverse Transcriptase and Integrase components. Transfer cPPT Central polypurine tract; recognition...γ-Retroviral transfer plasmid encoding a transgene, sgRNA, or shRNA of interest: The transgene sequence is...packaging and transduction are not encoded by the transfer plasmid, but instead are provided in trans by other...integrated into the host genome upon viral transduction. Most transfer plasmids available from Addgene were ...packaging, and transfer) are supplied by three types of plasmids, which are cotransfected into the 293T...cell line. γ-Retroviruses can transduce target cells, but the transduced target cell does not produce ...proviral DNA synthesis. Increases transduction efficiency and transgene expression. Psi (Ψ) RNA target...