...neuron which generates an action potential (Figure 1). This light-induced excitation can take place in ...channelrhodopsin activation to a small volume of brain tissue (~1 mm3). Current channelrhodopsins also need high-intensity...fiber-optic cables into the brain. Figure 1: With no blue light, the channelrhodopsin remains ... that determine membrane expression and plasma membrane localization of transmembrane proteins, like rhodopsins...capable of broad transduction of the nervous system which eliminates the need for intracranial injections. ... to perform in vivo optogenetic experiments. Intracranial injections of AAV are commonly used to deliver...channelrhodopsin-based optogenetics minimally-invasive, Frances Arnold’s lab and Viviana Gradinaru’s lab used machine...

...Collection Fluorescent tagging strategy Figure 1: Gene editing is the addition of a useful coding sequence...plasmid design strategy. A donor plasmid contains 1 kb homology arms matching the gene of interest, a ...Institute website at alleninstitute.org. References 1. Roberts, Brock, et al. "Systematic gene tagging using... copy. The coding sequence for GFP is transcribed and translated along with the gene, producing both a...exon of the gene to ensure it would be transcribed and translated. For an N-terminally tagged protein, ...destroyed by CRISPR/Cas9 during transfection. Making the double strand break and introducing the tag through...base pairs on each side, in fact. Later, when we transfect the cells, these large regions of homology enable...

...Popular gamma-Retroviral Transfer Vectors   Expressing your gene of interest pMKO.1 GFP (Plasmid #10676) ...GFP expression.  Cloning and expressing RNAi pMKO.1 puro (Plasmid #8452) RNA interference (RNAi) is an...information and a detailed protocol on how to use the pLKO.1 vector can be found here. Cre-Lox technology Puro....split-vector systems’ by separating viral genes and transgene expression across several plasmids. The most commonly...separate envelope and packaging plasmids as well as transfer plasmids. This concept ensures safe handling and...Gamma-retroviral vectors have a relatively high transduction efficiency, but in contrast to lentiviral vectors... are very versatile tools as they are able to transduce dividing and non-dividing cells. More detailed...

... biosensor, so that the (1) binding of calcium, (2) allosteric rearrangements of the sensor, and (3) subsequent... to a red fluorescent state only by simultaneous (1) user-controlled illumination with violet light and...fluorescent indicators. Nature protocols. 2006 Aug;1(3):1057-65. Resources on Addgene.org Addgene’s AAV...your expected range. For example, calcium in the ER lumen reaches the sub-millimolar range, much higher...are used to monitor calcium concentrations and transients in living cells and organisms. Unlike chemical...sensors available that will localize at the plasma membrane. GCaMP-X is a nucleus-targeted GCaMP6 with an ...an extra apoCaM-binding motif to prevent aberrant binding of the biosensor to endogenous calmodulin. Multiplexing...

...domain (Figure 1).  Figure 1: Examples of sites on antibodies used for conjugation. 1) Endogenous ...microscopy, flow cytometry, or spectrophotometry. The range of fluorophores available for antibody conjugation... choice for ELISAs.  Biotin Biotin is used in a range of antibody-based assays. Biotin itself is not visible...Endogenous carbohydrates added to antibodies post-translationally. 4) Modifications made to antibodies, such ...commercially conjugated antibodies available, that never guarantees the antibody you need will be conjugated, or ...Stephenson W, et al (2017) Simultaneous epitope and transcriptome measurement in single cells. Nat Methods 14:...

...current state (Green when off, dark when on). Figure 1: Photoswitchable MEK1 (psMEK1). Is it possible to ...Below are a few examples demonstrated in Zhou et al. 1. Testing kinase inhibitors Zhou et al examined the...the protein and pdDronpa are preserved. References 1. Zhou, Xin X., Linlin Z. Fan, Pengpeng Li, Kang Shen...Signal transduction pathways are a lot like cell phone networks. Protein kinases deliver messages to ..., and how long their signals last all impact transduction of the message. To study signaling pathways,... game of telephone, often the signal must be transmitted by intermediate messengers. Optical control of...signaling by using psMEK1 and an ERK kinase translocation reporter with a mRuby2 fluorescent reporter ...

...the other (Sandomenico et al., 2020). Figure 1: Comparison between the IgG antibody and scFv. ...intensity of a tagged protein. SunTag has two components: 1) a protein of interest that’s tagged with 10-24 copies...Application. Clinical and Developmental Immunology 2012:1–15 . https://doi.org/10.1155/2012/980250  Bates A,...without disulfide bonds, made by molecular evolution 1 1Edited by I. A. Wilson. Journal of Molecular Biology... the CAR facilitates signal transduction and release of cytotoxic granules from the T cell. When a CAR...., 2012, Bates and Power., 2019): rapid blood clearance, which is useful for imaging applications; better...small size (Bates and Power., 2019). Their rapid clearance from blood can be a drawback for therapeutic applications...

...glassware should be treated with a DEPC solution for 1 hour, and autoclaved to remove residual DEPC. Alternatively...more large, expensive ultracentrifuge.  Figure 1: An outline of the different steps in RNA extraction...extraction: twenty-something years on. Nature Protocols 1:581–585. https://doi.org/10.1038/nprot.2006.83 Additional...expense, and less of being left with a bunch of random solutions when all the spin columns run out. In... inherently not as stable as DNA- it is single stranded and its ribose groups are susceptible to hydrolysis...effectively isolate intact RNA, a fast, strong protein denaturant would be required- something that broke down ...Chirgwin and colleagues showed that a strong protein denaturant, guanidinium thiocyanate, did just this (Chirgwin...

... of the biotin-avidin interaction   Figure 1: Biotin is enriched in egg yolk while its binding ... substrate (Holden and Cremer, 2003). References 1. Chapman-Smith, A., & Cronan, J. E. “Molecular Biology...matrices.” Journal of Nanobiotechnology 13.1 (2015): 1–14. PubMed PMID: 25980573. PubMed Central PMCID: PMC4461904...detection by northern blotting.” Rice 7.1 (2014): 1–7. PubMed PMID: 26224555. PubMed Central PMCID: PMC4884002...purification of tagged transcription factors in mammalian cells and transgenic mice.” PNAS 100.13 (2003...and decarboxylases. These enzymes catalyze the transfer of carboxyl groups to organic acids to produce...lipogenesis, amino acid metabolism and energy transduction. Biotinylation is the general process by which...

... requires selection. These two steps are (Figure 1): The initial plasmid integration step (the so-called...antibiotic resistance marker from the vector (abR, Figure 1). The resolution step (“double crossover”) which leads... loss of a specific phenotype (using sacB, Figure 1, further discussed below).  Counter-selectable marker...Current protocols in molecular biology 106.1 (2014): 1-16. PubMed PMID: 24733238. Additional resources on...strong transcriptional terminator BBa_B1002 (from IGEM) upstream of the MCS to reduce transcriptional readthrough...rely on exogenous enzymes, require multiple transformation and plasmid curing steps, and can generate ...worked too. Rhamnose induction relies on the transcriptional activator rhaS. To allow manipulation of bacteria...

...succumb to the toxicity of CcdB.   Figure 1: Image from Bernard, P., et al. showing the pKIL 18...submit a guest blog entry of your own! References 1. Bernard, P. “Positive Selection of Recombinant DNA... plasmid ccdB killer gene”. Gene. 1994 Oct 11;148(1):71-4. PubMed PMID: 7926841. 4. Bernard, P., et al...poison, locking up DNA gyrase with broken double stranded DNA and ultimately causing cell death. ccdA, another...gene in frame with an MCS. E. coli that were transformed with the empty vectors expressed the ccdB gene... that ccdB Survival™ can be more difficult to transform, depending upon the specific plasmids being used...

... employing HDR. Ma et al. moved forward with gRNA 1 to conduct microinjection experiments in S phase zygotes...instead from the mother’s wild type allele (Figure 1). 72.4% of embryos carried two wild-type copies of...proceeded through NHEJ rather than HDR. Figure 1: Homology-directed repair in targeted zygotes proceeds...the embryos underwent repair not from the single-stranded oligonucleotide (ssODN) template supplied by Ma...maternal allele rather than the supplied single-stranded oligonucleotide (ssODN) template. Embryos also...off-target effects, but this advantage likely won’t transfer to all genes we’d like to target. In addition,...

...destabilize the transcription complex, initiating cleavage of the transcript.   Figure 1: A predicted... monkey virus constitutive RNA transport element lead to enhanced HIV-1 Gag expression in a context-dependent...prokaryotic terminators and deposited several with Addgene (1). Prokaryotic polyadenylation Although mostly thought...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...Hepatitis Virus Posttranscriptional Regulatory Element Enhances Expression of Transgenes  Delivered by ...

... two of the remaining viral proteins. Only Nsp11 ran bigger than expected, and Orf7b gave a lot of background...received so many requests (175 and counting) that we ran out of FedEx supplies at one point and had to go ...faster. Years of science, done in weeks Figure 1: Sending out 80 FedEx envelopes in a single day --...right) from the University of California, San Francisco. Manon Eckhardt and Melanie Brewer are on the ...

...Podcast breakdown (Time : Topic) 0:00 - 1:25 : Introduction 1:25 - 6:19 : Dr. Koeris' graduate work in...Novophage's successful transformation into Sample 6 and Dr. Koeris' own transition from graduate student...37-36:53 : Was It difficult for Dr. Koeris to transition from developing therapeutics to developing a ...

... DNA that were transcriptionally active in retinal cell types in mice. Figure 1: The four strategies...used four different design strategies: Strategy #1 (ProA): Create promoters with sequences upstream of...highest rate of targeted expression. Surprisingly, <1% of synthetic promoters replicated the expression ...mouse cell type specific transcription factor binding sites interspaced with random sequences.   Strategy...synthetic promoters. Legend: TSS: transcription start site. TFBS: transcription factor binding site. Image from...particular cell type is important not only for translational research, such as gene therapy, but also basic... using naturally occurring promoters to drive transgene expression, the Roska lab built synthetic promoters...

...yourself in a creative rut, read on!   Fig 1: Blugene using creative problem-solving in the lab...Blog Transferable Skills Guide: Problem-Solving Transferable Skills Guide: Teamwork Transferable Skills...feel they lack formal training in a variety of transferable skills. At Addgene we're helping to fill this...both helping researchers recognize their MANY transferable skills learned while working in the lab and ...Today, we’re highlighting an oft-underlooked transferable skill: creativity. I’ve heard quite a few scientists..., it’s clear that creativity is absolutely a transferable skill developed during PhD training. Creativity...problem. In a study outlined in Originals by Adam Grant, a creativity expert at Stanford conducted an experiment...

...various cyan fluorescent proteins is depicted in figure 1. Of note, the photobleaching rate does not depend ...to be used with the fluorescent protein. In figure 1 we provide an example of such a comparison between...the variants is comparable (lower panel of figure 1). To avoid changes in excitation power, it is important...fluorescent proteins in the same spectral class (Cranfill et al, 2016). This non-linear dependence on excitation...the conclusions reached at high power may not translate to real applications in which far less power is...

..., including those found at Addgene! Figure 1: Strategy for assembling the barcoded gRNA library...pool-clone them into the pAWp28 storage vector (Figure 1): Forward oligo: 5’- CACCGNNNNNNNNNNNNNNNNNNNGTTTGGGTCTTCGAGAAGACCTATTCXXXXXXXXC... He can be contacted at [email protected].   References 1. Cheng, Allen A., Huiming Ding, and Timothy K. Lu....expression of other genetic elements including transcription factors (Cheng et al., 2014) and microRNAs (.... Konermann, Silvana, et al. "Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex...

...functioning T cell repertoire (1). In this example, it was possible to transduce the T-cells outside the body...significant improvements in vision over a period of 1 to 3 years – the treatment appears to be both safe...research and science communication.     References 1. Gaspar, H. B., S. Howe, and A. J. Thrasher. "Gene...their mutations corrected, and the T-cells were transferred back into the infants. Almost all patients have...techniques may hold the key. In the simplest form, translating gene therapies from conceptual design to clinical...that carries a small, approximately 4.7 kb single stranded genome. AAV belongs to the family Parvoviridae...AAV vectors are also capable of infecting a wide range of cell types including but not limited to muscle...