We narrowed to 749 results for: Tro;
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TypeBlog PostPublishedJuly 11, 2019, 1:26 p.m....All three can introduce multiple base pair deletions, but CRISPR/Cas9 can also introduce point mutations...homologous recombination. A targeting vector is electroporated into embryonic stem cells and this will insert...joining, the newly inserted gene may have errors introduced to the nucleotide sequence during the DNA repair... in more detail in part 2 of this blog series) controls the cell types in which the conditional recombination...engineered mouse model allows gene expression to be controlled through a drug or small molecule that regulates..., Cre is fused to the ligand binding domain of estrogen receptor alpha (Esr1). Mice are fed or injected...
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Hot Plasmids and Viral Preps - January 2021
TypeBlog PostPublishedJan. 5, 2021, 2:15 p.m.... A fast blue-light inducible Cre for precise control of bacterial gene expression By Meghan Rego Recombinases...recombinases to manipulate genomes, scientists can control expression of their genes of interest. While recombinases...their activity can be fine-tuned and precisely controlled through the use of optogenetics. Optogenetic ...authors demonstrate that CRISPR-Casɸ is active in vitro as well as in human and plant cells. Casɸ...assay for mammalian cells. It's based on the introduction & resolution of two DSBs in a fluorescent reporter...serotype AAV5. Get the channelrhodopsin here Astrocyte specific tight affinity glutamate sensor from ...from the Sanford Boye lab with enhanced retinal tropism. Find the upcoming serotypes here ... -
Plasmids 101: Terminators and PolyA signals
TypeBlog PostPublishedMarch 31, 2016, 2:30 p.m.... affinity terminator may be desired for multi-cistronic constructs where high termination efficiency is...structure. It is thought that poly(A)s are used to control the cellular concentration of regulatory RNAs and... and may additionally act as a quality control mechanism to rid the cell of mis-folded RNAs. Eukaryotic...virus 40 late polyadenylation signal by using in vitro analyses. Molecular and Cellular Biology. 1992. ...Ther. 2008. PMID: 18627247 Zufferey R, Donello JE, Trono D, Hope TJ. Woodchuck Hepatitis Virus Posttranscriptional...Enhances Expression of Transgenes Delivered by Retroviral Vectors. Journal of Virology. 1999. PubMed PMID...Resources on the Addgene Blog Learn how the Promoter Controls Gene Expression Learn about New Cloning Techniques... -
The Materials Science of Optogenetics Experiments
TypeBlog PostPublishedSept. 17, 2015, 2:30 p.m....stimulation itself, the output of the laser must be controlled. As discussed above, each opsin variant has slightly...the A.M.P.I Master-9 Pulse Stimulator, which can control output of several lasers simultaneously, can be.... Pulse generation (TTL pulse) is a standard electronics protocol not specific to lasers or optogenetics...powerful alternatives to the Master-9 that only control one or two lasers are also available. Optimal stimulation...loss of laser power. More recently, wirelessly-controlled optogenetic implants have been reported by several...References 1. Gutierrez DV, et al. Optogenetic control of motor coordination by Gi/o protein-coupled vertebrate... Kim TI, et al. Injectable, cellular-scale optoelectronics with applications for wireless optogenetics... -
How-to: 5 Steps to a Great Panel Discussion
TypeBlog PostPublishedOct. 22, 2014, 1:57 p.m....do not let the panelists introduce themselves. I do a 1-2 minute introduction. If you chose good panelists...and will play a different role so her ability to control the event is what is important. Choose 4 panelists...logistics To make a really lively discussion, I strongly suggest asking the panelists not to use any slides...it (lapel mics are best). The moderator takes control It is the moderator's job to prevent anyone from...talking, so they will easily take 5 minutes to introduce themselves and, voila, 1/2 of your time is gone...publicity. Good panels do not waste time on long introductions. The moderator asks questions, calling on 1-... -
Pooled Library Amplifications
TypeBlog PostPublishedMay 30, 2024, 1:30 p.m.... of plasmids, they are generally electroporated into electrocompetent cells to ensure the highest possible...chemical transformations is less than that of electroporation, but an advantage is that chemical transformations...protocol, the size of the cuvettes your lab’s electroporator uses, etc. The DNA amount can be anywhere from...DNA in 100 μL of cells over four individual electroporations. The specifics of this can be based on the...5,000,000 30 bp At least 200 ng Liquid Electroporation Protocol (DOCX, 15.8 KB) Barcode SPLINTR... CRISPR Preparing Pooled Libraries NGS Quality Control for Pooled Libraries Resources on Addgene.org Addgene's... -
Expanded Access to Rabies Plasmids
TypeBlog PostPublishedFeb. 1, 2024, 2:15 p.m....SARS-COV-1 plasmids. Export control license process An export control license, once approved, allows...repository, including rabies plasmids, are export-controlled materials in the USA, where Addgene is located...applicable research project when purchasing export-controlled materials. Addgene will then apply for the license... Due to recent regulation changes, an export control license for rabies materials may be required for...application process. More information about export control licenses is available through the US Bureau of ... -
Isolating Genomic Regions of Interest with the CRISPR System
TypeBlog PostPublishedMay 30, 2019, 12:44 p.m.... they then confirmed by PCR. Subsequent mass spectroscopy studies allowed for the identification of proteins...Fujii Lab generated a transgenic mouse line and retroviral expression system for using this approach in ...analyzed, the Fujii lab also developed another "in vitro" form. In this form, the CRISPR complex, consisting...biotinylated gRNA, were successfully used in this "in vitro" form. Any tag system can be used to isolate genomic...expressed can cause facioscapulohumeral muscular dystrophy (FSHD), a disease for which there is currently... isolation of DNA fragments and chromatin by in vitro enCh IP technology using recombinant CRISPR ribonucleoproteins...as an RNA-guided platform for sequence-specific control of gene expression." Cell 152.5 (2013): 1173-1183... -
Antibodies 101: Producing Recombinant Antibodies
TypeBlog PostPublishedAug. 13, 2024, 1:15 p.m....Using a spectrophotometer is among the faster methods of quantification. Many spectrophotometers have an... you have multiple contaminating bands or a few strong ones, consider increasing the number of washes ... period of two weeks. After this time, quality control assays showed that the majority of antibodies are...references More resources on the Addgene blog Introduction to Antibodies Plasmid-based Recombinant Antibodies...Protein aggregation - Mechanisms, detection, and control. Int J Pharm. 2018 Oct 25;550(1-2):251-268. doi.../j.cocis.2014.10.002. Randolph TW, Schiltz E, Sederstrom D, Steinmann D, Mozziconacci O, Schöneich C, ... -
New Viral Vectors - Spring 2025
TypeBlog PostPublishedApril 29, 2025, 1:30 p.m.... channelrhodopsin in the retrograde serotype, and a dTomato under the control of the E6 regulatory element...pAAV-CAG-emiRFP670 AAV1 Control Kiryl Piatkevich New viral tool pAAV-S5E6-dTom-nlsdTom AAV1 Control Jordane Dimidschstein...Wilson New serotype AAVrg = AAV retrograde (also called AAV2-retro) Looking for more viral vectors? ... -
Deciphering the Mysteries of Behavior with Viral Vectors
TypeBlog PostPublishedSept. 17, 2019, 12:40 p.m....for this task. However, most lesioning methods destroy areas larger than intended and may unintentionally...to the neuronal networks involved in directly controlling muscle activity and coordinating movement, several...optogenetics to support these existing findings by controlling neurons in the mPFC with light. Activation of...inhibited by fear/anxiety. On the other hand, the control group preferred staying in the dark compartment...time the animal spends in the passive mode, the stronger is the depressive component of the animal behavior... such genetic study focuses on brain derived neurotrophic factor (BDNF). BDNF is a small protein involved...the same paper, the authors used lentivirus to introduce an shRNA to downregulate expression of miR124a... -
Bacterial Toxin-antitoxin Systems as Molecular Biology Tools
TypeBlog PostPublishedNov. 1, 2018, 12:35 p.m.... to introduce multiple genetic modifications in the same strain. MazF expression is under control of the... a GFP cassette, and introduced site-directed mutations, all without introducing unwanted selection markers...delivery vector can be integrated into the genome to introduce the genetic modification(s) contained within it...EcoRI and Bsp6I in Escherichia coli in 1995 when introduction of restriction-modification systems onto plasmids... able to monitor lacZ activity over 48 hours in vitro without antibiotic selection. In a subcutaneous ... -
Analyzing CRISPR Editing Results with ICE from Synthego
TypeBlog PostPublishedMay 8, 2018, 1 p.m....derived from the control trace file. On the plot, the green (edited sample) and orange (control sample) lines...cells, genomic DNA from both edited and unedited (control) populations is PCR-amplified and Sanger sequenced...determined by comparing the edited trace to the control trace. In the ICE algorithm, potential editing ...summary table. You can also use your browser’s Control+F or Command+F functionality to search for a particular...disagreement between the non-edited wild type (control) and the edited sample in the region around the...difference. Traces The Traces tab shows the edited and control, non-edited Sanger traces in the region around ... -
5 Reasons to Use Reddit for Science Communication
TypeBlog PostPublishedOct. 20, 2016, 2:30 p.m....unproductive conversations with internet trolls (urban dictionary for trolling: The art of deliberately, cleverly...to write our AMA submission to avoid rousing the trolls. 3. The AMA process was simple and well organized...while it can be anonymous, I can always block the trolls. It seemed like I would really have to open myself...moderators were on top of any comments that were at all trollish. Kudos to the moderators for keeping the quality...discourse. They even advised me on how to alter my intro to reduce the number of potentially hostile posters... the term “implicit bias” which is apparently a troll trigger phrase these days). Redditors ask serious... -
Getting the Most from Your Lentiviral Transduction
TypeBlog PostPublishedApril 7, 2016, 2:30 p.m....approach complete with freely available positive controls. Routinely test your cell line for mycoplasma ...freezing virus in a dry ice/ethanol bath or liquid nitrogen prior to storing. Know your target cells Labs ...These binding events can be impaired by the electrostatic repulsion of the negatively charged cell with..., A., Garnier, A. “Downstream processing of oncoretroviral and lentiviral gene therapy vectors.” Biotechnology...conditions on the production of lentiviral and retroviral vectors.” Human Gene Therapy Clinical Development...Cronin, J., Zhang, X.Y., Reiser, J. “Altering the tropism of lentiviral vectors through pseudotyping.” Current...Cleghorn G., West M.J., Wei MQ. “The significance of controlled conditions in lentiviral vector titration and... -
Twenty Years of Sharing: Expanding Accessibility
TypeBlog PostPublishedNov. 21, 2024, 10:47 p.m....Optogenetics Guide γ-Retrovirus Guide Protocols Agarose Gel Electrophoresis Bacterial Transformation... direct shipments weren’t able to clear import control. Partnering with a distributor allowed us to share... How to Design Primers for PCR Agarose Gel Electrophoresis Inoculating Liquid Bacterial Cultures Restriction...Restriction Digest Analysis We also introduced guides — practical primers to help users get started ...we launched the Addgene YouTube channel to add a strong visual component to our educational resources. ... systems to allow for easier access to export-controlled items by streamlining permissions and paperwork... -
Career Insights: Technical Support Specialist
TypeBlog PostPublishedNov. 9, 2017, 2 p.m....different scientific fields, and knowledge of how to troubleshoot problems at the bench. Specifically, roles in...loads of questions, and I also had to do some troubleshooting as advised by the technical support person....specificity of this antibody?” You will also help troubleshoot products, give general experimental guidance...the world easier. You should also demonstrate a strong willingness to help others with their problems,... initial contact, but is less suitable for troubleshooting or issuing replacement products. In order to...careers are open to experienced professionals with strong interest in the technical aspects of science and...work includes reviewing customer data sets, troubleshooting protocols, analyzing information, and solving... -
Multicolor Animals: Using Fluorescent Proteins to Understand Single Cell Behavior
TypeBlog PostPublishedMarch 5, 2019, 1:08 p.m....field of view. Tetbow can be introduced in vivo either by electroporation of plasmids, or using AAV vectors...type of cell labeling technique involves the introduction of a transgene construct containing fluorescent...cells, the expression level of these XFP is not strong enough for all applications. To resolve this, similar...use in Drosophila melanogaster and is under the control of Flp recombinase. The XFPs in Flybow are membrane-tethered...entire organism or a specific tissue. XFPs are introduced using a plasmid construct called Zebrabow-GateDest...Additional resources on the Addgene blog Read an introduction to live cell imaging Learn more about the origin... -
Viral Vectors 101: Integrase-Deficient Lentiviral Vectors
TypeBlog PostPublishedMay 13, 2025, 1:15 p.m....CRISPR/Cas9 to cells for genome editing, and introducing inducible systems into cells, among many other...Desfarges & Ciuffi, 2012). One family of viruses, retroviridae, require integration for their life cycle to... during production (read our blog post on the retroviral life cycle for a more detailed description). ...concerning in clinical settings, as evidenced by retroviral gene therapy trials for X-linked severe combined...immunodeficiency virus type 1 integrase mutants retain in vitro integrase activity yet fail to integrate viral DNA...lessons from provirus and non-integrated DNA. Retrovirology, 1, 13. https://doi.org/10.1186/1742-4690-1-...resources on the Addgene blog Viral Vectors 101: The Retroviral Life Cycle Quick Guide to All Things Lentivirus... -
Plasmids 101: Common Lab E. coli Strains
TypeBlog PostPublishedNov. 7, 2014, 2:56 p.m....Stbl4 Electrocompetent cells for cloning and storage of unstable DNA and lentiviral/retroviral vectors...Allows uptake of large plasmids hee High electroporation efficiency supE44 (glnV44) Suppression ...cloning and storage, blue/white screening, leucine auxotroph. F- endA1 recA1 galE15 galK16 nupG rpsL ΔlacX74...For cloning into and storage of lentiviral and retroviral vectors or cloning or repeated sequences with... -
CRISPR/Cas9 FAQs Answered!
TypeBlog PostPublishedMarch 13, 2014, 4:08 p.m....Engineering Using CRISPR/Cas Systems has expanded out of control. To further consolidate these FAQs, Le Cong worked...gRNAs with the Cas9 nickase which will together introduce cleavage of the target site with efficiency similar...on each side flanking the change you'd like to introduce. We use Ultramer oligo from IDT (non-PAGE purified...although this might still be okay if you are introducing a selection marker gene. The homology arms should...ideally less than 10 bp away if possible. For introducing small mutations (<50 bp) or a single-point mutation...around 100–150 bp total homology. The mutation is introduced in the middle, giving 50–75 bp homology arms....double digestion. As a successful insertion will destroy the BbsI sites, a double digest should discriminate... -
Genetically-encoded Sparse Cell Labeling - A SPARC of Innovation
TypeBlog PostPublishedMay 21, 2020, 1:15 p.m....system along with precisely truncated attP sites to control recombination of the gene construct. A predictable...gene. Effector genes, any gene that you want to control the expression of using the SPARC system. The effector...rest of the SPARC components and expression is controlled by the upstream recombination outcome. Find ...Find SPARC plasmids at Addgene To more precisely control the amount of labeling, you can alter the attP site...effector position, as well as to optogenetically control the depolarization of R2 neurons using CsChrimson...genome engineering in flies Learn more about controlling gene expression in flies Resources on Addgene.org... -
Plasmids 101: What is a plasmid?
TypeBlog PostPublishedApril 2, 2020, 6:17 p.m....scientists and plasmid lovers - serves as an introduction to plasmids. Plasmids 101 will provide you with...plasmids contain an origin of replication (which controls the host range and copy number of the plasmid)...the lab are usually artificial and designed to introduce foreign DNA into another cell. Minimally, lab-... that scientists can purify and study it in a controlled setting. Read more: Plasmids 101: Protein Tags...environment Produce enzymes that will make specific, controlled changes to an organism’s genome (genome engineering...guide also contains multiple protocols and troubleshooting tips to make plasmid usage as simple and straightforward... -
Hot Plasmids and Viral Preps - July 2021
TypeBlog PostPublishedJuly 27, 2021, 1:15 p.m....mimic aspects of organ morphology and physiology in vitro and have emerged as a powerful model for understanding...for doing gene targeting, reporter generation, controllable gene knockouts, and inducible gene inactivation...design. B) Strategy for leak-free transcriptional control. C) Iterative transduction workflow overview. Image...SL-insensitive fluorophore (LSSmOrange) as an internal control and a SL-sensitive fluorophore (circularly permuted...the cpGFP without altering that of the internal control (Fig 1). Because this method relies on the fluorescence...-500nM range) and a high specificity for SLs in vitro, and they further demonstrated that when tested ... -
New and Upcoming Viral Vectors - Spring 2019
TypeBlog PostPublishedMarch 26, 2019, 1:40 p.m....activity in the brain was measured with implanted electrodes. The Archon1 voltage reporter allows a way to...cre-dependent version at Addgene! Fluorescent protein and control AAV These fluorescent protein expression vectors...interneurons. These vectors can also be used as the control vectors for experiments encoding a fluorescent-... Addgene in the next 6 months, pending quality control. For an estimate on availability date or to be ... and serotype you’d like to be notified about. Controls Plasmid Serotype Name 114472 AAV5, AAV8 ...Plasmid Serotype Name 119036 AAV1 pAAV-hsyn-flex-Voltron-ST Chemogenetics Plasmid Serotype Name ... -
In Vivo Biotinylation of Bacterial Fusion Proteins
TypeBlog PostPublishedJan. 25, 2018, 2:09 p.m.... Many thanks to our guest blogger Jon Backstrom. Jon Backstrom, Ph.D is a biochemist in the Vanderbilt...This post was contributed by guest blogger Jon Backstrom, a biochemist in the Vanderbilt Eye Institute ...oligomerization of biotinylated proteins on avidin can be controlled easier than formation of GST dimers. Avi-Tag ... of added biotins per molecule cannot be well-controlled. The lack of site selectivity typically diminishes...relatively low efficiency of biotinylation, at least in vitro. Our solution was to exploit the best of both worlds... -
A Practical Guide to Optimizing AAV DIO and FLEx Vector Expression
TypeBlog PostPublishedNov. 2, 2021, 1:15 p.m....exhibit profound differences in tropism between cell types (astrocytes, microglia, neurons, etc.) and ...can not recombine with each other. When Cre is introduced to the system it binds one pair of lox sites,...your cells. Some promoters, such as CAG are very strong and may require less viral particles or relatively...or glial fibrillary acidic protein (GFAP) for astrocytes it is important to test it in the brain region... S (2013) Analysis of Transduction Efficiency, Tropism and Axonal Transport of AAV Serotypes 1, 2, 5, ...and 5 Display Differential Efficiency and Cell Tropism after Delivery to Different Regions of the Central... -
New Neuroscience Tool: The iGluSnFR3 Glutamate Sensor
TypeBlog PostPublishedApril 7, 2022, 1:15 p.m.... inserted GFP. After characterizing iGluSnFR in vitro, showing that it is selectively activated by glutamate...higher expression levels in bacteria and produces stronger fluorescent signals upon sensing glutamate in ...variants differing in kinetics, affinity, and ∆F/F0. Introduce the two variants in three different membrane-targeting...iGluSnFR3 have higher expression levels and produce stronger fluorescent signals upon sensing glutamate using...addition to the original PDGFR TMD, the researchers introduce iGluSnFR3 in two additional anchoring domains ...induction to express soluble protein CAG: Generic strong promoter. Formulated as AAV-DJ for neuronal expression... -
Hot Plasmids - January 2023
TypeBlog PostPublishedJan. 31, 2023, 2:15 p.m....CRISPR-Csm: A novel RNA targeting system by: Susanna Stroik The Doudna lab’s recent work characterized a new...heat-inducible lentiviral plasmids here! Wu Y, et. al. Control of the activity of CAR-T cells within tumours via...chemically stable tool for microscopy by: Susanna Stroik Microscopy techniques have been moving at the speed...microscopy applications such as super resolution, electron, and expansion microscopy. hfYFP is not only brilliantly...denaturing conditions, high temperatures, and other chaotropic conditions better than most FPs. The group also...expansion microscopy as well as correlative light and electron microscopy and observed high fluorescence resilience... -
Hot Plasmids - December 2019 - CRISPR pooled library, LipoGlo, CRISPRa, and Voltage Imaging Tools
TypeBlog PostPublishedDec. 10, 2019, 1:58 p.m....plasmid deposit please sign up here. A retroviral mouse CRISPR knockout library Article contributed...phenotype, and this depends on the method used to introduce the gRNAs into cells. Most CRISPR pooled libraries...issue, Sarah Teichmann and colleagues created a retroviral CRISPR knockout library using gRNAs designed ...designed by the Kosuke Yusa Lab. This retroviral library was used to transduce T helper type 2 (Th2) cells from...differentiation factors as the screen readout. This retroviral mouse CRISPR pooled library can be used for screening...concentration of ApoB-LPs has been shown to have a strong correlation with cardiovascular disease and indirect...