...’s fused to the C-terminus of DsRed.T3. See Table 1 for a summary of Rosella’s excitation and emission...fluorescence Rosella emits based on its localization. Table 1: Summary of Rosella’s excitation and emission spectra... variants are available from Addgene. References 1. Rosado, C., Mijaljica, D., Hatzinisiriou, I., Prescott...single fluorescent biosensor. Scientific Reports,5(1). doi:10.1038/srep12397. PubMed PMID: 26215030. PubMed...Autophagosomes are relatively easy to identify: double-membraned structures containing undigested cytoplasmic contents...stands for protein) associates with autophagosomal membranes, so tagging it with GFP allows for tracking the...spectra. Component Excitation Emission pH Range DsRed.T3 488, 543*, 568 nm 587 nm ~4.9 - 9 ...

...within the mitochondrial matrix.     Figure 1: A chart with MoClo kit recommendations for different...Traditional Biology.” ACS Synthetic Biology, vol. 5, no. 1, American Chemical Society (ACS), 4 Nov. 2015, pp...., vol. 4, no. 9, American Chemical Society (ACS), 1 May 2015, pp. 975–86. Crossref, https://doi.org/10.1021...Yeast Mitochondria.” BMC Biotechnology, vol. 17, no. 1, Springer Science and Business Media LLC, 13 Nov. ...Editing in Plants.” BMC Plant Biology, vol. 20, no. 1, Springer Science and Business Media LLC, 23 Apr. ...Arabidopsis Mutants.” The Plant Journal, vol. 106, no. 1, Wiley, 25 Mar. 2021, pp. 8–22. Crossref, https://...Cloning Platform.” Scientific Reports, vol. 9, no. 1, Springer Science and Business Media LLC, 12 July ...

... produced by transfecting the following four components into 293T cells. Figure 1: The key components...protein. A single Nanoblade particle can be loaded with 1-4 different gRNAs. Viral envelope proteins To alter... between ~50-70% for these four cell types (Table 1). Additionally, hiPSCs and mouse BM cells still behaved...Myd88  ~50% Human HSCs  Myd88  ~50% Table 1: Nanoblades editing efficiencies of primary cells. Comparing...delivery include plasmid transfection, RNP electroporation, and viral transduction; but these methods aren...they can then fuse with the target cell membrane, and transfer their CRISPR cargo into the zygote. As ...Repair (HDR), CRISPR activation or repression, transgenic mouse line creation, and in vivo gene editing...

...inducible gene expression. Transgenic and knockin mice As the name suggests, transgenic and knockin mice contain...their genome. While transgenic mice have the new gene construct incorporated randomly into the genome, knockin...Conversely, outbred strains were originally derived from random crosses between mice of uncharacterized backgrounds...several types of genetically engineered mice: transgenic mice, knockout mice, and mice with conditional...construct inserted at a specific site. To generate a transgenic mouse, microinjection of DNA or infection with...Non-homologous recombination incorporates this DNA randomly into the mouse genome. In contrast, knockin strains...animal for gestation. Nucleases that induce double-stranded DNA breaks, such as ZFN and TALEN, can used to...

...in FRET are: 1. Donor fluorophore excitation by absorption of a photon 2. Energy transfer from the excited... located within 1-10nm of each other and be oriented appropriately for energy transfer via dipole-dipole...decision easier. 1. Müller, Sara M., et al. "Quantification of Förster resonance energy transfer by monitoring...diseases and even assist in drug discovery. References 1. Baird, Geoffrey S., et al. "Circular permutation ...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...

... hybridization experiments. A recent publication [1] from the Sánchez lab describes the plasmids and protocols...deposit) are described in the Sánchez lab publication [1] so that the students performing these experiments...can easily investigate in planarians. References 1. Alice Accorsi, Monique M. Williams, Eric J. Ross,...Proceedings of the National Academy of Sciences 100.suppl 1 (2003): 11861-11865. PubMed PMID: 12917490. PubMed...remove one strand of the siRNA and are directed by complementarity to the remaining single stranded siRNA ...activity or their translation disrupted through physical blocking of the translation machinery. In the...WISH) for visualizing expression of genes. Double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) to...

...C2c1, C2c2 and C2c3 (short for Class 2, candidate 1, 2, or 3). C2c1 and C2c3 are related to Cpf1, but ...lacks the ability to cleave the RNA target.  Table 1: Comparison on Common CRISPR Enzymes Name  Enzymatic...Blog Posts Applications of Cas13a   Figure 1: Using Cas13a as a diagnostic tool. A pool of DNA ... This system, dubbed SHERLOCK (depicted in figure 1) has been used to differentiate strains of Zika virus...contributed to the update of this post. References 1. Abudayyeh, Omar O., et al. "C2c2 is a single-component...Recombinase Polymerase Amplification (RPA) or Reverse-transcription RPA (RT-RPA), respectively. Amplified nucleotides...

...and plant transformation. Hortic Res 7, 152 (2020). https://doi.org/10.1038/s41438-020-00390-1 Additional...in all other plants, anthocyanins.   Figure 1: An adult plant expressing RUBY. Photos by Andrew ... be collected from heterozygous individuals. Only 1/4 of the descendants of the heterozygous plants, therefore...Selectable transgene markers for plant transformation fall into two main categories: antibiotic/herbicide...presence of a transgene instead, which can’t be done if your selection method kills the transgene free individual...Arabidopsis seed following floral dip transformation next to an untransformed seed from the same plant beginning...resistance genes usually involves killing the untransformed seedlings or callus (undifferentiated plant ...

... the example shown in Figure 1, large sequences corresponding to exons 1 and 3 of the targeted gene have...knock out exon 2 of a given gene.   Figure 1: A knockout targeting vector designed to insert a ... the Neomycin resistance (NeoR) gene between exon 1 and 3 of the targeted gene. Upon recombination, the... mechanism to accurately repair harmful double stranded breaks, in which nucleotide sequences are exchanged...selection marker HSV-tk is used to select against random recombinants.   Design your targeting construct...selecting for cells in which the construct has randomly integrated into the genome, a negative selection...step will kill off many of the cells that have randomly integrated all or large portions of the plasmid...

...events by HDR can be generated more efficiently by (1) improving editing accuracy by preventing re-editing...specifically incorporate homo- or heterozygous mutations. 1. Increasing HDR accuracy with CRISPR/Cas-blocking ...targeted alleles when gene editing.   Figure 1: Techniques to improve editing efficiency.   But...provided by blocking mutations. Essentially, you:  Step 1 - Introduce the blocking mutation together with the...developing novel iPSC-based model systems.     References 1. Cong, Le, et al. "Multiplex genome engineering using...com­plex is very efficient at introducing double stranded breaks (DSBs) into genomic DNA in many cell types...homologous DNA repair template, such as a sin­gle-stranded oligodeoxynucleotide (ssODN), which contains the...

...milliliter can be detected – in the zeptomolar range Time: ~1 hour Equipment required: 37°C water bath and... to other technologies like CRISPR. Figure 1: An overview of the general schematic of CRISPR/Cas...nearly single-molecule sensitive. In the case of HIV-1 and SARS-CoV-2 detection, AIOD-CRISPR without preamplification... Not mentioned in the paper, likely approximately 1-2 hours Equipment needed: Mini PCR machines by DIY-Bio...Sensitivity: – 110 femtomolar  Time: Not mentioned, likely ~1-2 hours Equipment needed: PCR machines/Heat blocks...the zeptomolar range while One-step-SHERLOCK has a sensitivity in the femto-aptomolar range. For more details...are designed, each binding to one strand of the reverse-transcribed target sequence that are present close...

...photosensitizer used in this work is composed of two parts: 1) an activatable protein (the FAP - fluorogen activating...reaching greater than 75% photobleaching after just 1 minute of exposure. MG-2I-dL5** experienced minor ...photosensitizers in the comments below!   References 1. He, J., Wang, Y., Missinato, M. A., Onuoha, E., Perkins... encoded photosensitizer. Nature Biotechnology,24(1), 95-99. doi:10.1038/nbt1175. PubMed PMID: 16369538...these experiments because developing embryos are transparent, so it’s easy to watch development in real time...

...Brainbow-1 and -2. The general structure of Brainbow-1.0 is retained in Brainbow-3.0, but with mOrange2, EGFP...the Brainbow mouse neural labeling system. Brainbow-1 and -2 have been further refined to create Brainbow...subsequent recombinations cannot occur. While the Brainbow-1 system employs Cre-mediated deletion, the design of...antibodies specific to each fluorophore. Third, Brainbow-1 and Brainbow-2 each contained a “default” state; for...Rheenen J, Simons BD, Clevers H. Cell. 2010 Oct 1;143(1):134-44. PubMed. Drosophila Brainbow: a recombinase-based...proteins, which are directly trafficked to cell membranes. Membrane trafficking of farnesylated derivatives enables... virus post-transcriptional regulatory element (WPRE), commonly used to increase transgene protein levels...

...how scientists connect on Twitter that I realized 1) a lot of scientists are using social media professionally... but my consistent writing for the Addgene blog (~1-2 blog posts per month) and use of Twitter and LinkedIn... have tips for using social media as a scientist. 1. "5 Ways Scientists SHOULD Be Using Twitter" podcast...of interest. Hopefully through these links, I’ve: 1) convinced you social media can positively impact ... to research potential future employers and to arrange informational interviews. 4. “Tips for Scientists...

...intended.)  Steps to clear writing Let’s use Figure 1 as our example. Remember, our goal is to use words...information as sighted readers.    Figure 1: An image from AAV Purification by Iodixanol Gradient...are they hoping to get from it? The image in Figure 1 appears on our protocol page for AAV Purification ... share with the rest of the workshop.  For Figure 1, Addgene chose this alt text: "The left panel is a...resources on the Addgene blog Five Steps to Writing Grant Proposals to Grad Students Writing Scientific Manuscripts...

... vectors are: 1) the potential generation of replication competent virus [usually HIV-1]; and 2) the potential...systems have been developed over the years based on HIV-1; however, the 1st-generation vectors are never used...which has been shown to repress expression of HIV-1 LTRs (more information is available here). 2nd vs...considered. Q2: Can I use lentiviral transfer vectors for transient transfections? A2: Technically, yes. Will ...produced by co-transfecting three essential ingredients: the lentiviral packaging vector, a transfer vector including...specifically for transient expression (find some useful backbones here) or from viral transduction. While not...3rd-generation transfer vectors apart? A3: Addgene defines 2nd- or 3rd-generation transfer vectors based...

...perform, at minimum, two transformations after a ligation: 1. Control Transformation containing the ligation...competent cells. For most standard cloning, you can transform 1-2μl of your ligation reaction into competent ...well, so let’s go over the details step-by-step. 1. Digestion Set up restriction digests for your insert...recipient plasmid : insert ratio” of approximately 1:3. Since the number of base pairs for each varies,...result in few colonies on both plates (unsuccessful 1), in a vector alone plate with many more colonies ...contamination from uncut plasmid. 4. Transformation Transform your ligation reaction into your bacterial... how many colonies you should expect in the transformation due to background re-circularization and contamination...

.... Table 1 provides an overview of pooled library protocols you can use as a reference. Table 1: Examples...backbone might be more difficult to transform.    Figure 1: Factors to consider when creating a...etc. The DNA amount can be anywhere from 10 ng to 1 μg of DNA. It often correlates with the size of the...libraries, chemical transformations can also be used. Efficiency for chemical transformations is less than that...for your transformation(s). The amount of competent cells you need total and per transformation may vary...the manufacturer’s protocol for transformation and expected transformation efficiency, or more specifically... coverage, since this will help decide what transformation efficiency you need to achieve. Most pooled...

...2 (CRY2) and calcium and integrin-binding protein 1 (CIB1) proteins. The goal of both groups was to create... the Sata lab is composed of two fusion proteins: 1) the genomic anchor - an inactive, dead Cas9 protein...combination. The optimized LACE system consisted of: 1) CIBN-dCas9-CIBN, where CIBN is the N-terminal fragment.... crassa) (Kawano et al., 2015). Nicknamed paCas9-1 and consisting of the fusion proteins N713-pMag and...fold-induction of Cas9 activity (16.4-fold). This paCas9-1 light-inducible system was able to recognize the same...flCas9). When triggered by blue light (470nm), paCas9-1 induced indel mutations via NHEJ (frequency of 20.5%...background activity of the system by modifying paCas9-1 using nMagC714 instead of nMagHigh1-C714, generating...

...2021).   Table 1: Properties of modern NIR FPs     NIR FP Ex, nm Em, nm ECa, M-1cm-1 QYb, %   Molecular...expression level. State-of-the-art FPs (listed in Table 1) were optimized for efficient binding of endogenous...previously reported widely used dimeric iRFPs (Table 1). In general, the monomeric state of a probe leads...iRFPs in cellular and molecular brightness (Table 1).  NIR reporters and biosensors NIR reporters can ...dDetermined as effective NIR fluorescence in transiently transfected live HeLa cells with no supply of exogenous...lamps are the best for working in this spectral range. You can also check that no filters and/or cold ...and after normalization to fluorescence of co-transfected EGFP. e Originally reported as a monomer16, IFP2.0...

...total dose between 1 x 1011 and 5 x 1011 vg per animal (i.e., 10 and 50 µL of vector at 1 x 1013 vg/mL titer... and 1 x 1012 vg*. Most systemic capsids are easy to produce in high titer, but higher doses (>1 x 1012...titers than AAV-PHP.B (e.g., 1 x 1011 vg/mouse), AAV-PHP.eB can transduce a greater number of neurons ...intravenous gene transfer throughout the non-human primate brain. Nature Nanotechnology, 1–11. https://doi.org...endothelial cell transduction throughout the central nervous system. Nature Cardiovascular Research, 1(4), Article...capsids facilitates enhanced transgene expression in the macaque CNS. Med, 4(1), 31-50.e8. https://doi.org...before using it in an application.     Figure 1: AAV toolkit for targeted gene delivery to the brain...

...attract fish for them to prey upon (Figure 1). Figure 1: The sea anemone's red color can be used ...synthetic biology. Journal of Biological Engineering, 12(1). https://doi.org/10.1186/s13036-018-0100-0 Murakoshi...expression in living cells. Scientific Reports, 9(1). https://doi.org/10.1038/s41598-019-48604-4 Salih...chromoproteins are just now being developed for a broad range of biological inquiries.  What is a chromoprotein... a gene encoding a blue chromoprotein, after transforming them into your cells, you can simply look for... plasmids (meffRed, eforRed, asPink, spisPink, scOrange, fwYellow, amilGFP, amajLime, cjBlue, meffBlue...Image: Murakoshi et al., 2019. ShadowR is an orange light-absorbing chromoprotein that Hideji Murakoshi...

... different steps and materials required for each [1, 2]. This protocol [3] provides, in exquisite detail...follow him on Twitter @derekpsimonphd.    References 1. Gutierrez DV, et al. Optogenetic control of motor...manipulation of neural circuits. Nature protocols. 2012;7(1):12-23. doi: 10.1038/nprot.2011.413. PubMed PMID: ...freely moving mice. Neuroscience research. 2011;70(1):124-7. doi: 10.1016/j.neures.2011.01.007. PubMed ...tissue, placement above the site of stimulation guarantees that the majority of the opsins will receive ...tangled. Furthermore, to give the animal the full range of motion, a commutator allows the patch cable to...

...on-target and off-target editing efficiency Figure 1: Effects of (A) double and (B) single base pair gRNA-target...that Cpf1 can tolerate mismatches at gRNA positions 1, 8, 9, and 19-23. Accordingly, the 3’ end of the gRNA...Most gRNAs directed low-frequency Cpf1 cleavage at 1-12 off-target sites; in contrast, SpCas9 may cleave...fits into the genome editing landscape. References 1. Kim D, Kim J, Hur JK, Been KW, Yoon SH, Kim JS. Genome-wide...pattern opens up the possibility of directional gene transfer, analogous to traditional restriction enzyme cloning... cloning. Sticky-end mediated gene transfer would be particularly helpful for targeting non-dividing cells...gene delivery due to their low immunogenicity and range of serotypes allowing preferential infection of ...

...genome replication and transcription of negative strand RNA viruses.   References 1. Buchholz, Ursula J....which are required for replication and transcription (Figure 1). All viral genes are removed and replaced...negative strand RNA viruses: The power of reverse genetics. Springer Berlin Heidelberg, 2004. 1-41. PubMed...cells that stably express T7 (e.g. BSR T7/5 cells) (1). We hope that minigenomes will greatly enhance our...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 ...

...by mating with Cre-expressing mice. Figure 1: (A) Schematic of the Cre-dependent SPH construct ... alpha (aCaMKII; excitatory neurons) and Synapsin-1 (pan-neuronal) promoters (Figure 1b), and sgRNAs can...ubiquitous expression of sgRNA(s) and mCherry (vector 1) with neuron-specific expression of dCas9-VPR (Figure...Neuron-specific expression is mediated by the synapsin 1 (SYN) promoter, which was superior in its ability ...engineered combinations of transcriptional activators to induce gene transcription at genomic sites targeted...p65-HSF1 transcriptional activator to each genome-bound dCas9 (see Figure 1a for the transgenic construct...different untranslated or coding regions. Typically, each gene isoform has a distinct transcriptional start...

...cells need three different plasmids to produce AAV: 1) the RepCap plasmid which provides the AAV replication...plasmid. This whole process takes ~3 hours to complete: 1 hours hands-on time and 2 hours for the qPCR run and...about AAV production and purification! References 1. Aurnhammer, C., Haase, M., Muether, N., Hausl, M.... repeat sequences. Human gene therapy methods, 23 1, 18-28. PubMed PMID: 22428977. 2. Choi, V.W., Asokan...proteins to process a double-stranded intermediate into the single-stranded genome that's then packaged...viral replication; and 3) the transfer plasmid which encodes a transgene of interest that’s packaged into...XmaI restriction sites). Another way is to transform AAV transfer plasmids into bacterial strains, like NEB...

...against the limitations of the assay.      Fig. 1: A schematic representation of Sybr Green chemistry...Methods Mol Biol. 2018;1768:143-160. doi: 10.1007/978-1-4939-7778-9_9. PMID: 29717442. Arya M, Shergill IS...amplification of Plasmodium falciparum multidrug resistance-1 gene (pfmdr1). J Parasitol. 2011 Oct;97(5):939-42....gamma receptor genes FCGR3A and FCGR3B. PloS one, 10(1), e0116791. https://doi.org/10.1371/journal.pone.0116791...pipetting error or an incredible discovery. The range of RQ values used to call copy numbers should be...fluorescent dye that works by binding to all double-stranded DNA, while Taqman uses an intercalating dye, with...confident determination of copy number, albeit within a range of RQ values as noted above.   In both applications...

...Protocols Publication describing them here. References 1.Ran, F. Ann, et al. "Genome engineering using the CRISPR-Cas9...plasmid post transfection. The 2A "self cleaving" peptide causes the ribosome to translate Cas9-2A and ...component of most CRISPR systems and generates double strange breaks at target sites 5' of the 5'NGG3' PAM sequence...backbone. This particular backbone can be used to transfect a variety of mammalian cells and can be modified...are some additional features. The first and most imporant is that, upstream of the Cbh promoter, this plasmid...can be used to test for SpCas9 expression post transfection by western blot. Overall, this is a versatile...

...discovered such as Kaede and PS-CFP2 (see Table 1). Table 1: Properties of Selected Irreversible Photoactivatable...) by mutating Threonine 203 to Histidine (T203H) (1). wtGFP normally contains a mixed population of neutral...in fluorescence when illuminated with blue light (1). Similar to PA-GFP, scientists later developed red...fixation conditions to preserve cellular structures (0.5-1% osmium tetroxide). These conditions destroy most ...with extraordinary molecular detail.   References 1.Patterson, George H., and Jennifer Lippincott-Schwartz...is believing." Annu. Rev. Biomed. Eng. 10 (2008): 1-38. PubMed PMID:18647110. Additional Resources on ...fluorescent PA-FPs by performing several rounds of random mutagenesis screening for enhanced RFP variants...

...dollar contracts behind closed doors. Fig. 1: The mythical land of negotiations.    You negotiate...feel they lack formal training in a variety of transferable skills. At Addgene, we've set out to fill this... by highlighting that researchers learn many transferable skills while working in the lab and by offering...where you might need some help. Today in our transferable skills guide: Negotiation. As a grad student...I was negotiating with my PI on how to spend my grant money (Can I attend this conference?) and with my...Hawaii?). So how can you approach improving this transferable skill? Negotiation as a zero sum game Some ...is the 6th year grad student frustrated that the brand new grad student keeps using the machine? Let...

...and Therapeutic Purposes. Annu Rev Virol. 2014 Nov;1(1):427-51. DOI: 10.1146/annurev-virology-031413-085355...personalized medicine. References and further reading 1. FDA approves gene therapy for blindness 2. Gene Therapy... Med Biol. 2014; 801: 687–693.  doi:  10.1007/978-1-4614-3209-8_86. PubMed PMID: 24664759. PubMed Central...and they had been shown to to transduce the retina but only transiently. However new viral vectors capable...adenovirus (AdV) and lentivirus (LV) are known to transduce retinal cells, AAV quickly became the vector of...due to its considerable immunogenicity and its transient expression. LV results in long term expression... in dividing and non-dividing cells due to its (random) integration into the genome, and its large DNA...

..., 10(1). https://doi.org/10.1038/s41467-019-12449-2 Pupo, A., Fernández, A., Low, S. H., François, A.,...therapy of human diseases. Signal Transduction and Targeted Therapy, 9(1). https://doi.org/10.1038/s41392...NGS) to identify genes of interest. Figure 1: Overview of in vitro AAV CRISPR screens. Created ...certain experiments. AAV can be safely handled at BSL-1, while lentivirus requires a BSL-2 setup. This requires...vivo starts with transducing cells with the CRISPR library in vitro before transplanting them into mice....time. This reduces the chance of integration at random locations that could cause deleterious effects....portions of the AAV genome can integrate at double-stranded break points caused by CRISPR-Cas9 editing. The...

...identification of proteins associated with the IRF-1 locus. Figure 1: Schematic of enCHIP. Image from Fujita... interest, the promoter region of the transcription factor IRF-1. Immunoprecipitation, using an antibody...References Campbell, Amy E., et al. "NuRD and CAF-1-mediated silencing of the D4Z4 array is modulated ...dCas9 has focused on transcriptional activation using a fusion to a transcriptional activator such as VP64...repression of transcription through binding a promoter region to inhibit association of transcriptional activators...2018). Furthermore, the Fujii Lab generated a transgenic mouse line and retroviral expression system for...Tapscott Lab identified regulators of DUX4, a transcriptional regulator that when inappropriately expressed...

...10.1038/s41551-024-01227-1. doi:  https://doi.org/10.1038/s41551-024-01227-1   CHARM: A compact epigenetic...spacer proteins ending with a GFP nanobody (Figure 1). This enrichment streamlines the purification and...highly-visible beads on the cryo-EM grid.     Figure 1: MagIC-cryo-EM for sample enrichment and structure...single-particle analysis of PRC2 with RvLEAMshort (1:6 molar ratio) with 10 minutes of glutaraldehyde crosslinking...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...

... export license applications for VSV and SARS-COV-1 plasmids. Export control license process    An export...charged.  Timelines  We have offered VSV and SARS-COV-1 plasmids under this application process since May ...where Addgene is located.  The export license, if granted, allows us to ship rabies plasmids and viral vectors...

...new combinations of PE protein components (Figure 1), an altered epegRNA delivered with an enhanced pU6cm...amplification of cargo, and heat treatment.  Figure 1: Left: recombined prime editing tools PE2max-NC and...conformational dynamics of the motor protein kinesin-1, the Twelvetrees Lab took advantage of the self-labeling..., Craggs, T.D., & Twelvetrees A.E. (2024) Kinesin-1 is highly flexible and adopts an open conformation...NC: nucleocapsid RNA chaperone, RT: reverse transcriptase. Right: T1 tomato plants that inherited an edited...Cultured rat hippocampal neuron expressing pan-membrane ASAP5. Solid blue circle marks the initiation ...via the Kv2.1 targeting motif as well as a pan-membrane construct without the Kv motif. Altogether, if...

...markers in the panel. This complexity index ranges from 0-1, with a value of “0” indicating those markers...detector for that fluorophore. For example, in figure 1, the FITC signal is spilling over into the primary...spectra to use for compensation.     Figure 1: The emission spectra and filters for FITC and PE ...completely distinct from one another, and a value of “1” indicating those markers are identical and completely...

... here! Podcast Breakdown (Time: Topic) 0:00-1:00 : Intro 1:01-3:48 : Kingdom Kwapata's background and ...Michigan State University 9:36-10:29 : Kingdom's transition back to Malawi and Lilongwe University of Agriculture...-14:53 : Kingdom's current projects producing transgenic crops 14:54-23:44 : Follow up Kingdom's PhD projects... transgenic crops...

...introduce their new friend…  Fig. 1: Meet...well, frankly, we're not sure yet!   This as-yet–...narrowed it down to three choices.   Fig. 1: Lin, Abi, or Blottie? Which one suits me best?   ...choices), we'll be in touch with you soon with some brand-new Addgene swag!   Last month, we introduced you...out to you and received a plethora of options, ranging from Affie to Yncredibody. After an agonizing week...

... Fig. 1: Barcoded TF library was packaged in lentiviral particles and used to transduce human embryonic... J. et al., A transcription factor atlas of directed differentiation. Cell. 2023. 186 (1) 209-229. doi...TF library, with each cell receiving only 1 TF expression construct. Cells were cultured following a differentiation...Transcription factors (TFs) act as the gate keepers for gene expression, turning transcription on and... regulatory network. The transcription atlas and library The transcription factor library The human...approximately 1,800 TFs, with >3,500 unique isoforms transcribed from these sites. The Zhang lab generated a comprehensive... contribute to differentiation, the Zhang lab transduced human embryonic stem cells with the viral TF ...

...) vector (Figure 1). Everything else outside the ITRs gets left behind.    Figure 1: Recombinant AAV ...reduces rAAV productivity but increases transgene expression. Sci Rep, 7(1), 5432. https://doi.org/10.1038/s41598...containing transcription factor binding sites can increase transgene expression and transduction efficiency...as the gene that they package. In a transfer plasmid, the transgene is flanked by two ITRs, and the ITRs...modifications can drastically affect gene transfer efficiency and transgene expression (Zhou et al. 2017). For...these mutations can affect transduction efficiency and subsequent transgene expression. Replacing the ...nucleotide sequences that flank the ends of the single stranded DNA genome of AAV in the native virus. In an AAV...

...118). Humana Press. https://doi.org/10.1385/1-59259-301-1:99 Resources on Addgene.org Addgene antibodies...and yes, we have an antibody for that!   Fig. 1 - Diagram of His-tagged protein purification work ...blot and immunofluorescence. GST  Glutathione S-Transferase (GST) is a 26 kDa tag which can be isolated with...

...setting out of the lab (coffee, lunch or beer) and in 1:1 conversations. This creates a level of familiarity... other posts in our Science Mentors series: Part 1: "What Makes a Good Mentor?" and 6 More FAQs About... opportunities to learn and practice successful grant writing, lab budgeting, equipment acquisition, public...

...cross-neutralization occurs in the P.1 and B.1.351 variants tested. Image from Garcia-Beltran et al., 2021 with permission...for the wild-type. However, variants such as the P.1 (Gamma variant) and the B.1.351 (Beta variant) were... tested at the time of this study. Figure 1: Neutralization levels against spike pseudovirus decreases...vaccines differed depending on the variants (Garcia-Beltran et al., 2021). Antibodies from individuals who ...represent lineages and variants that are actually transmitting out in the world” says Balazs. “By combining...quantified in terms of luminescence. Image from Garcia-Beltran et al., 2021 with permission.   Mutations ...with various pseudotyped virus. Image from Garcia-Beltran et al., 2021 with permission.   Difference ...

...directed against human transcription factors such as PAX7, PAX6, AP-2 alpha and ISLET-1. In addition, we distribute...Monoclonal Antibody Research Institute dedicated to: 1) developing new ways of generating antibodies, for...light chains and two heavy chains. The ends of the branches contain the antigen binding sites and are highly...

...2013) Analysis of Transduction Efficiency, Tropism and Axonal Transport of AAV Serotypes 1, 2, 5, 6, 8 and...(Figure 1). This results in one loxp site and one lox2272 site on either end of the transgene. Since loxp...cause of this spurious expression?   Figure 1.  Cre-recombinase binds to either pair of lox sites...recombination and can exhibit recombination levels as high as 1-3%. In light of this finding, we now include guidance...Vectors Pseudotyped with Viral Capsids from Serotypes 1, 2, and 5 Display Differential Efficiency and Cell...loxp pair, and flips the transgene into the correct orientation for transcription. Upon recombination one...flank a transgene (homologous pairs) or used different scrambled sequences to flank the transgenes (non-...

... and reproducibility of a study’s findings. Table 1: Biased Genotyping Options Technique Sequence Information...sequencing doesn’t tell the whole story Figure 1: Schematic representations of the indicated unbiased...types Must be verified with cell-based method IDLV 1% Cell-based Programmable, can detect DSBs in live ...magazine.scienceforthepeople.org/.     References 1. Fu, Yanfang, et al. "High-frequency off-target mutagenesis...next-generation sequencing." BMC genomics 15.1 (2014): 1. PubMed PMID: 25409780. PubMed Central PMCID: PMC4246457...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...

... cleaved out of the RNA transcript by endogenous processing enzymes (Figure 1). To ensure this tRNA processing... mRNA trans-splicing. A cis-splicing vector served as positive control (cis-ctrl). v1, vector 1; v2, vector...mRNA trans-splicing dual AAV vectors for (epi)genome editing and gene therapy. Nat Commun., 14(1), 6578...or nuclear expression of BFP2.     Figure 1: CROPseq-multi uses two sgRNAs with internal barcodes...RNA polymerase III transcript, the sgRNAs were encoded on the lentiviral minus strand in the 3’ LTR. These...HeLa cells transduced with SARS-CoV-2 nucleocapsid protein (Plasmid #141391-LV [+N]) or membrane protein ...high microglia transduction efficiency for the indicated ssAAV5s. Figure adapted from Serrano et al. 2023...

...nonstructural proteins 1 to 16, spike proteins, envelope small proteins, and membrane proteins. Although ...give your research more visibility.    Figure 1: Illustration of SARS-CoV-2 from Philippa Steinberg...Humana Press, pp 331–338 https://doi.org/10.1007/978-1-59745-181-9_23 Additional resources on the Addgene...glycoproteins and other proteins that require post-translational modification may not be easily expressed in ...sequencing as a first pass QC. The lab team manually transforms, innoculates, and performs DNA minipreps for ...