.... Find the Dolcetto lentiviral prep here Control AAV Control AAV allow researchers to not only interpret...many of our control plasmids in new serotypes, as well as a new Cre dependent GFP control plasmid for ...mediated neuronal control. The advantages of optogenetics lie in the spatiotemporal control offered by the...for their own experiments. To this end, we offer control AAV in smaller aliquots of 20ul for serotype testing...also offered as AAV1 (along with AAV PHP.eB, AAV Retrograde, AAV2, AAV5, AAV8, and AAV9). Once activated,... Addgene in the next 6 months, pending quality control. For an estimate on availability date or to be ... AAVrg pAAV-hDlx-GqDREADD-dTomato-Fishell-4 Controls Plasmid Serotype Name 114471 AAV2 pAAV-Ef1a-fDIO...

...type SOX2 with either a SOX2-VP16 (strong activator) or SOX2-HP1 (strong repressor) fusion protein, the authors...elavl3/HuC), microglial/macrophage (mpeg1.1), and astrocytic (gfap).This toolbox adds new neuronal tools to...to express the transcription factors from a polycistronic cassette. However, it was unknown whether variations...variations in gene expression from these polycistronic cassettes could lead to comparable experimental...Woltjen Lab compared different variants of the polycistronic cassettes and discovered reprogramming discrepancies...variation in the Klf4 isoform (KLF4s or KLF4L). Polycistronic cassettes that contained KLF4s had overall diminished...reprogramming and global gene expression patterns. Polycistronic cassettes that contained the KLF4L isoform had...

... and destroyed by CRISPR/Cas9 during transfection. Making the double strand break and introducing the ...individual proteins under endogenous regulatory control. But even with the advent of CRISPR/Cas9 technology... in human cell lines. Furthermore, the quality control necessary to ensure the edited cells are behaving...the tag through homology directed repair To introduce the fluorescent tag to the cells, we used CRISPR/Cas9...with the cut, we trick the cell into using the introduced donor plasmid as a repair template. This results...and the donor plasmid into the cells we use electroporation, a technique that briefly destabilizes the ...Figure 3: Inducing the double strand break and introducing the tag. Created with BioRender.com. Plasmids...

...system to modify your target DNA, you first electroporate a linear donor DNA substrate (either dsDNA or...and SbcCD nucleases from digesting linear DNA introduced into the E. coli. Exo: Exo is a 5’→3’ dsDNA-dependent...Expression of lambda red recombination genes. Electroporation of substrate DNA and outgrowth of bacteria....dsDNA recombineering substrates to avoid errors introduced during PCR. ssDNA Substrate ssDNA substrates ...that avoid MMR activation.  The first way is to introduce a C/C mismatch at or within 6 base pairs of the...1% or 25-50% when activation of MMR is avoided Introduce C/C mismatches or silent changes in wobble codons...these will be discussed in more detail below).  Controlling expression of Red proteins is critical for minimizing...

...would like to introduce the edit. Finally, you must stipulate what sequence to introduce, which is done...guide’s recognition sequence is destroyed via mutation. By introducing a mutation in either the PAM or...Identify where in the genome you would like to introduce your knock-in. Then choose which Cas enzyme to...region of a gene, make sure that the PAM edit you introduce is a silent mutation, so you don’t accidentally...to your culture media prior to and after Cas9 introduction increases HDR, just make sure to remove the ...homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLIFE. (2014)...efficient homology-directed repair and termperature-controlled genome editing. Nat. Com., 8(2024), (2017). 10.1038...

...-GFP AAV9-X1.1 Controls Edward Boyden New serotype pAAV-hSyn-EGFP AAV PHP.eB Controls Bryan Roth New...CN3905) AAV PHP.eB Controls Jonathan Ting New viral prep pAAV-CaMKIIa-EGFP AAV1 Controls Bryan Roth New ...minBG-SYFP2-WPRE3-BGHpA (Alias: CN3044) AAV PHP.eB Controls Jonathan Ting New viral prep AiP13038 - pAAV-AiE0743m...minBG-SYFP2-WPRE3-BGHpA (Alias: CN3038) AAV PHP.eB Controls Jonathan Ting New viral prep AiP12408 - pAAV-AiE0600m-minBG-SYFP2...pAAV-AiE0600m-minBG-SYFP2-WPRE3-BGHpA (Alias: CN2408) AAV PHP.eB Controls Jonathan Ting New viral prep AiP13863 - pAAV-AiE0888m_C4...minBG-SYFP2-WPRE3-BGHpA (Alias: CN3863) AAV PHP.eB Controls Jonathan Ting New viral prep AiP13905 - pAAV-AiE0441h...New serotype pAAV-Ef1a-DIO mScarlet AAV5 Controls Karl Deisseroth New serotype pAAV_hSyn-PdCO-mScarlet-WPRE...

...emission Before we design your first panel, we will introduce some background on the color spectrum and the ... photons are absorbed by matter, they promote electrons within atoms to a higher energy state. This process... called excitation. After a short period, the electrons revert to a lower energy state, whereby a photon...those of absorbed photons. When photons excite electrons of a fluorophore, they can do so at a range of...by the cytometer. Brighter fluorophores have a stronger signal. Table 1: Degrees of brightness for a few...absorbed and then emitted, which creates a relatively stronger — or brighter — signal.   Figure 2: Excitation... real-life advantage of a brighter fluorophore? Strong brightness makes it easier to distinguish a signal...

...deliver the mini-dystrophin gene to a mouse model of muscular dystrophy. While mini-dystrophin expression levels... to deliver a 6 kb mini-dystrophin gene to a mouse model of muscular dystrophy. Three different splice... and expression of the full-length transgene in vitro and in vivo (Allocca et al., 2008). Shortly after...levels were lower than expression of wild-type dystrophin in normal muscles, mice treated with the overlapping...achieved ~80% of muscle cells expressing mini-dystrophin.  3. Hybrid  Overview: The hybrid approach combines... can be from one of the transgene’s endogenous introns or comprised of non-endogenous sequences, such ...Gene Therapy of mdx Mice With Large Truncated Dystrophins Generated by Recombination Using rAAV6. Molecular...

...commercial isothermal assembly master mix. Introduction of the strong transcriptional terminator BBa_B1002 ...decide which path to take. A quick and easy way to introduce a mutation in the chromosome is to disrupt expression...called allelic exchange. Here, a mutated allele introduced from a conjugative plasmid replaces the chromosomal...frequently requires considerable optimization to ensure strong counter-selection. Anecdotally, there is also a... evolved specifically for toxicity would allow stronger counter-selection than one merely co-opted toward...rhamnose- and arabinose-inducible promoters. Tight control of toxin expression is absolutely necessary, because...Proteobacteria. We placed the toxin genes under control of a rhamnose-inducible promoter, though Khetrapal...

...colors you like. Controls and validation Can’t forget the controls! The necessary control experiments will...from Addgene), or be prepared to run some extra controls and validate the antibody yourself. If you can... we’ll walk you through the necessary types of controls for a multiplex experiment.  Check for non-specific...using a different antibody.  Prepare a positive control Prepare and label a sample that expresses your ...naturally or by overexpression, for a positive control. You can also confirm the secondary works as expected...S., Diaz, M. F. P., & Yong, W. H. (2019). An Introduction to Performing Immunofluorescence Staining. In....2018488117 Additional Resources on the Addgene blog Introduction to Antibodies Which Fluorescence Microscopy ...

...If you're just getting started, check out our Introduction to PCR.) But if you're ready to go beyond the...tips on probe chemistries, primer optimization, controls, and analysis.  As a rule of thumb, you can assume...experimental parameters (neighbor stacking energies, loop entropy effects, cation concentrations and a temperature...side and a 5% negative error on the other will introduce unwanted variation into your data. Try different...protocol may have to be re-optimized.  While troublesome, multiplexing your reference gene set can save...the fold change of each sample compared to your control. But as we said previously, just because it’s used...assumes the amplification kinetics of the target and control genes are fairly equal, an assumption that is not...

...tail vein or retro-orbitally Gauge: 27–30 Volume: < 0.2 ml Skill required: beginner (retro-orbital) to ...restrainer or anesthesia Tail vein or retro-orbital 27–30 < 0.2 mL Beginner (retro-orbital) to intermediate (tail...2020). A retrospective analysis for different routes of administration in Mice-Percutaneous Retro-Orbital...vein injections.” However, they can also be given retro-orbitally, behind the eye. IV injections are used...needle in the correct vein without collapsing it. Retro-orbital injections are also thought to be less stressful...decision Hopefully this post has provided a good introduction to common injection routes in mice! When considering...

...recombineering machinery are first electroporated with pCas9. Then, pCRISPR is introduced along with an oligonucleotide... in a bacterial strain introduced to the population as methods of introduction. Engineering the bee gut... of knockdown and developed new, more tightly controlled, Tet responsive promoters to avoid leaky activation...systems differ on how exactly the CRISPRi locus is introduced (Tn7 in Gram-positives and ICEbs1 in Gram-negatives...CRISPR system targets these genomic locations, and introduces double strand breaks, the bacteria are unable...This approach provides a more granular level of control where individual bases can be modified. Cytidine... that could be fused to dCas9 and efficiently introduce cytosine-to-thymine substitutions in target sequences...

...super-resolution microscopy or electron microscopy. Since its introduction in 2015 by the Boyden Lab, ExM...expand evenly in all directions, aka isotropically. But achieving isotropic expansion depends on how well the...2010). How do you overcome this resolution limit? Electron Microscopy (EM) is one option, but the equipment...much to be done about that.  Second, expansion introduces distortions. The goal in expansion is for your...variation, your samples can tear or expand unevenly, introducing distortions in your final images. It is possible...a deeper understanding of neuronal synapses, centrosomes, parasites, and more (Bertiaux, et al. 2021; ...resources on the Addgene blog Antibodies 101: Introduction to Immunofluorescence  Antibodies 101: Multiplex...

...type of edit you need to introduce highly specific? Perhaps you need to introduce a single nucleotide modification...McDade and significantly updated in 2022 by Susanna Stroik.  The advent of CRISPR/Cas9 has made it easier ...right for your unique experiment. Here, we will introduce you to the wide array of Cas9s (many available...Knock-out cells or animals are engineered by introducing Cas9-mediated frameshift mutations in the gene...interest. The genomic target for Cas9 and the introduction of this mutation can be any site early in the...and knockouts Some experiments may require the introduction of a mutation at a specific site e.g., deleting...Synergistic Activation Mediator complex, generally the strongest activator, consists of a dCas9-VP64 fusion and...

...1998). Regulatory elements within the ori also control copy number and stability, which influence the ...which consist of a plasmid’s ori and all of its control elements, from broad host range plasmids can be...Hypomycrobium X, Legionella pneumophila, Methylophilus methyltrophus, Methylococcus methanolicus, Methylosinus trichosporium...hydrophila, Agrobacterium tumefaciens, Alcaligenes eutrophus, Azotobacter vinelandii, Brevibacterium methylicum...Klebsiella aerogenes, K. pneumoniae, Methylophilus methylotrophus, Moraxella spp., Mycobacterium aurum, M. smegmatis...Agrobacterium tumefaciens, A. rhizogenes, Alcaligenes eutrophus, Enterobacter sp., Erwinia amylovora, E. carotovora...spp., Legionella pneumophila, Methylophilus methylotrophus, Myxococcus virescens, M. xanthus, Proteus ...

...protein. With this control set, you should see your target protein IP’d in the positive control sample but not... Basics of Western Immunoblotting.) For mass spectrometry, elute in a urea-containing lysis buffer. Alternatively...permanently altering the structure of the protein.  Controls As with any experiment, it is critical to include...include proper positive and negative controls for an IP. When possible, perform the IP in parallel with ...interest and a sample that does not. Common negative controls include knockout cell lines or tissues that do... not in the negative control, confirming that the capture antibody is functioning as expected. In order...perform parallel IPs with beads only and an isotype control antibody. Alternative applications Oftentimes, ...

...your cell of interest: AAV tropism and route of administration AAV tropism is defined by the viral capsid...FRT, with AAV vectors are common approaches to control transgene expression by restricting expression ...regulatory elements such as promoters and enhancers, multicistronic vectors, and post-translational regulatory ...Combinatorial strategies can also be used for retrograde tracing: visualizing neurons that received information...region. In the example shown in Figure 1, injected retrograde AAVs at one location result in opsin expression...viral vector related blog posts Learn more about retrograde AAVs Resources on Addgene.org Read our AAV ...guide Learn more about our viral service Find retrograde AAVs at Addgene ...

...updated Nov 3, 2022 by Susanna Stroik. You’ve designed your gRNA and introduced it into your target cells ... of your target site is. If you are trying to introduce a homozygous mutation, it’s helpful to know how...knockout cell line is desired, any mutation that introduces a frameshift in the coding region of interest... sequence clones to verify that the deletions introduced are out of frame but finding clones with deletions....   Validating knock-in mutations You’ve introduced a donor template along with your gRNA and Cas9...your desired edit is. Large knock-ins Are you introducing a large insertion, something 20 base pairs or...restriction enzyme sites? Never fear! You can introduce a silent, passenger edit to serve as a proxy for...

..., that strongly binds the glycoprotein, avidin or a similar protein, streptavidin. This strong interaction...antibody based affinity purification and mass spectrometry approaches. Recently however, scientists have...classic antibody based affinity purification/mass spectrometry methods.  BioID: a promiscuous biotin ligase...ascorbate peroxidase (APEX), used as a genetic tag for electron microscopy, could be used for efficient proximity...of hydrogen peroxide. This radical reacts with electron-rich amino acids such as tyrosine on neighboring...exogenous biotin allowing for more precise temporal control.  Find plasmids for TurboID and miniturbo Split-BioID...Stephanie S., et al. "Directed evolution of APEX2 for electron microscopy and proximity labeling." Nature methods...