...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...

...) 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...

... 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 ...

...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 ...