...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...

...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...

... 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 ...

...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...

...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...

...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...

...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...

...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...

...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...

...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...

...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 ...

... 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...

...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 ...

...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...

...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...

...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...

... 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...

... to neuroscience. In fact, precise light-based control of biological processes is highly useful in other... to be optimized by Tabor’s lab, making light-controlled gene expression in E. coli easier and more robust...engineered to deliver precise, consistent, and controllable pulses of light. Complicated patterns of light...new pathways with sophisticated gene expression controls. Most cell types do not respond to low-intensity...two systems can be coexpressed for sophisticated control of gene expression. An E. coli light-sensitive...from this publication at Addgene. Multichromatic control of gene expression in Escherichia coli. Tabor JJ...

...need to ensure you have a bead-only control alongside your input control. Elute the DNA after washing and..., which uses formaldehyde or UV light to create strong but reversible bonds. This will keep the protein...of histone modifying proteins, which bind quite strongly to DNA, crosslinking may not be necessary. This...Enzymatic digestion, while gentler on the sample, introduces bias due to the selective nature of the cutting...Antibodies 101: Polyclonal Antibodies Antibodies 101: Introduction to Antibodies Resources on Addgene.org ...

...sequence NGG TTTV PAM site preserved? Usually Destroyed Yes, Cpf1 cleaves 5' of the protospacer Multiplexing...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  ...showing that Cpf1 can cleave an array of 4 crRNAs in vitro and when expressed in 293 cells. This allows for...a GFP tag. Four weeks post-infection, there was strong expression of Cpf1 and GFP in the targeted brain...effective method with multiple applications (in vitro, in vivo, transfection, viral transduction) for ...