...Viral Vectors 101: An Introduction to AAV Viral Vectors 101: The AAV Retrograde Serotype Viral Vectors... more than one.  Most AAV serotypes have broad tropism (the ability of a virus to enter a particular cell... cell type), but there are differences in tropism between animal species or even strains of the same species...well in a rat or non-human primate. Likewise, in vitro efficacy doesn’t always translate to in vivo efficacy...remember that some serotypes can be transported retrogradely and/or anterogradely along the axon. Others,...site or circuit can occur. Promoters The promoter controls how the gene of interest will be expressed; it...or inducible.  Ubiquitous promoters  These often-strong promoters offer the most flexibility for expression...

... PCR-introduced mutations can make alternative approaches more favorable if you want to introduce a point...highly versatile technique that can be used to introduce specific nucleotide substitutions (or deletions...approach can be used in conventional cloning (to introduce or remove restriction sites), in mapping of regulatory...substitution of key residues), and in SNP analysis (to introduce naturally occuring SNPs in a plasmid context)....genome editing. Tailored mutations are commonly introduced to endogeneous DNA through homology-directed ... Mutagenesis In brief, point-mutations can be introduced to plasmids using primers (with the desired mutation...green) amplify the plasmid template (blue), and introduce a restriction site “A*” (in addition to the “A...

...plasmids: Luc (intronless; CMV-LUC2CP/ARE and Luc-I (intron-containing; CMV-LUC2CP/intron/ARE). The Luc-I...all-optical electrophysiology A major challenge in the field of all-optical electrophysiology is designing...field with an improved tool for electrophysiology, minus the use of electrodes. The Cohen lab has made the...Luc-I intron is a chimeric β-globin/immunoglobulin intron that has been commonly used in constitutive splicing...an effort to obtain even more precise temporal control of gene knockout or activation, the c-terminal ...). This system provides users greater temporal control over CRISPR/Cas9 mediated genome modification and...Optogenetics is a powerful tool that utilizes light to control and monitor individual living cells in order to...

...muscular atrophy.   Type of Virus Pros Cons Retrovirus Stable Gene Expression Broad Tropism ...the best one for your experiment. Retro and lenti viruses Retroviruses are a family of viruses that stably...The most well-known retrovirus is HIV. Lentiviruses are a genus of the retroviral family that can infect...which means they do not stimulate a strong immune response when introduced into an organism. This feature ...straightforward across applications, while electroporation of a human, for example, isn’t a reasonable...types of viruses used in mammalian research: 𝛄-retrovirus, lentivirus, adenovirus, and adeno-associated...the need for envelope disassembly. Both lenti and retro viruses can accommodate a cargo up to approximately...

...range (tropism). Pseudotyping is only done on enveloped viruses such as lentivirus, retrovirus, and rabies...et al, 2020). Because retrovirus is closely related to lentivirus, many retroviral envelope proteins have... a few cell types.  If you’re looking for more control in the cell types your lentiviral vector will infect...particles.   Why pseudotype? Lentiviruses and retroviruses typically target immune cells with their wild-type...viruses to infect neurons. Besides altering the host tropism of your virus, there are many other reasons when.... Image from Crawford et al., 2020.   Broad tropism: VSV-G pseudotyping A commonly used envelope protein...stem cells and neurons. However, VSV-G’s broad tropism can be undesired for in vivo studies since there...

...blotting is a loading control. Pro-Tips: 1. It’s all about the MOI. In retrospect I used a way too high...express CAs9 and are included as a control (WT). Vinculin is a loading control. 3. In terms of making the...undigested control. Sometimes the PCR products can look smeared, so having an undigested control can help...those formed from control (GFP gRNA-targeted) BRAF sites (lanes 3, 4) were used as controls. Duplexes were...but that nobody talks about. After months of troubleshooting, those people who did not tell you that essential...Cas9 line that’s “most efficient at editing” to introduce the gRNA, but I just randomly chose one of my ...and BRDN0001146266) ( 76479-LV and 76480-LV) or control GFP gRNA (BRDN0000561167) (80034-LV) were individually...

... application for OptoNBs takes us in vitro: light-controlled affinity purification of unmodified proteins...light-based control of signaling pathways. This is particularly exciting because using light to control signaling...  Option #2 may sound like fiction, but light-controlled protein purification is now a reality thanks ..., and their use for protein purification and controlling cell signaling. Key components of OptoNBs OptoNBs... put AsLOV2 because here it was less likely to destroy the nanobody’s function than if inserted into the...insertion into loops 1 and 6 resulted in light controlled binding, but with opposite effects: insertion...insertion site on light-induced binding.  Light controlled protein purification with OptoNBs The first application...

... when using auxotrophic selection Of course, there are some drawbacks to using auxotrophic markers as ...maintained within the yeast cell. These elements control not only the number of plasmids found in each cell...unstable and may be lost during budding. Yeast Centromere plasmids (YCp): These are considered low copy...incorporate part of an ARS along with part of a centromere sequence (CEN). These vectors replicate as though... The copy number of these vectors can also be controlled if specific regulatable elements are included...yeast Historically, scientists have utilized auxotrophic selection rather than antibiotic selection when...insensitivity of yeast strains to some antibiotics. Auxotrophy is defined as the inability of an organism to...

...possible, include positive and negative controls. Positive controls typically include cell lines or tissues...Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure...Towbin et al., 1979, Burnett et al., 1981).  Introduction to western blotting  In a western, proteins ... Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is the first step of a western. ...immobilized onto a membrane, usually composed of a nitrocellulose or polyvinylidene difluoride (PVDF) in a step...step called protein transfer. While both nitrocellulose and PVDF are commonly used, PVDF tends to be slightly...validated applications, pay close attention to the controls listed on the vendor’s website. Typically, the...

...www.janelia.org/open-science/voltron-and-positron.  Both Ace2N-mNeon-A122D and Voltron2 are available as Cre-dependent...33714753. Voltron2 voltage sensors By Brian O’Neill New viral vector preps expressing the Voltron2 voltage...performance.      Figure 4: Structure of Voltron or Voltron2 conjugated to a JaneliaFluor dye emitting... SARS-CoV-2 research New viral vector preps of Voltron2 voltage sensors AAV vectors for targeting microglia...membrane protein (Plasmid #141386-LV [+M], negative control) were immunoblotted against Anti-SARS-CoV-2 Nucleocapsid...also immunoblotted against vinculin as a loading control (right panel). Image from Addgene. These antibodies..., are now available! Building on their earlier Voltron indicators (a fusion of Ace2N and HaloTag, Figure...

...subunit (RPP30) as a control for normalization purposes are being used. Quality Control Mycoplasma Our 293T...virus preps are titered and subjected to quality control by Addgene before being distributed to customers...production protocols, titering methods, and quality control are described below. AAV Production AAV distributed...probes targeting the ITR elements and an internal control of known titer (protocol modified from Lock et ... April 2016) also performed by ddPCR.  Quality Control Addgene ensures high quality viral vectors by optimizing...production protocols and performing rigorous quality control (QC). The specific QC experiments performed varies...preparations are subjected to polyacrylamide gel electrophoresis (PAGE) followed by silver staining or SYPRO...

...and other proteins controlled by GAL4 and ZIF268. To demonstrate independent control of gene expression...a GAL4/estradiol controlled transcription factor and a ZIF268/progesterone controlled transcription factor...transcription factor 1 controls expression of transcription factor 2, which directly controls expression of a...TRIM21 recruits ubiquitin and the proteasome to destroy antibody-bound pathogens. Clift et al. found that...can deliver these components together using electroporation. In these bulk cell experiments, Clift et al...no stress or damage upon TRIM-Away, and the electroporated cells re-adhered to the culture plate within...chemically inducible approaches to provide temporal control, this new system, Fkbp/Frb Inducible Recruitment...

... rather blunt instrument for controlling CRISPR. To bring greater control to Acr activity as a biological...Listen to this episode of Hot Plasmids! Optogenetic control of CRISPR-Cas9 by CASANOVA Article contributed ...protein domains, they have developed a system to control CRISPR-Cas9 activity using blue light (Bubeck et...opens up the potential for spatial and temporal control in many genetic targeting and editing experiments...electrical activity in the brain by inserting electrodes into the brain. But this process is time consuming...photostable, and is compatible with optogenetic control. The researchers also showed that Archon1 is functional...cloning podcast segment Recently, the Grefen lab introduced the “2in1” vector cloning system - a Gateway ...

...described the first light-activated tool for controlling neuronal activity, channelrhodopsin-2 (ChR2),...scientists have developed many optogenetic tools to control cellular activity with exquisite precision using...light. Optogenetics, or light activated tools, can control everything from electrical activity to gene expression...BioRender.com.   Optogenetic tools Types of control We will discuss three types of tools: light-gated...hyperpolarize the cell (Mattis et al., 2012). Bicistronic vectors combine excitation and inhibition by ...Some tools turn on and off rapidly with light to control precise timing of the perturbation. Other tools...intra-cellular signaling cascades. Optical methods for controlling GPCR activation have been developed to selectively...

...efficient tools to introduce your gene of interest into cells. Unlike gamma-retroviruses that can only infect...Q1: Are retroviruses and lentiviruses the same thing? Can I package lentivirus with retroviral packaging...Lentiviruses are part of the retroviral family. What scientists often refer to as “retroviruses” are technically ...technically gamma-retroviruses - another, separate member of the retroviral family. While both lentiviruses and ...Addgene Depositor (and lentiviral expert) Didier Trono's lab webpage explains how lentiviral particles are...and gamma-retroviruses use the same genes for packaging (that's gag, pol, and env), the isoforms of these...LTRs) are different. As a result, lentiviral and retroviral packaging vectors are not interchangeable. While...

...produced in high-titer retroviral stocks generated by the transfer of a retrovirus into the packaging cell...are interested in using lentiviral vectors to introduce your favourite gene into your favourite cell line...CRISPR/Cas9 components, and fluorescent sensors. Retroviral vector background Until the development of viral...cells, labs use viral vectors and particularly retroviral vectors because they target a broader range of...into the genome is more efficient. The first retroviral vectors used were derived from the Moloney Murine...MoMLV, lentiviruses, a separate genus of the Retroviridae family including human immunodeficiency virus...infect non-dividing cells is not restricted to in vitro cell culture as lentivirus-derived vectors are capable...

...inefficiently our edits were being introduced. Although we were able to introduce the desired mutations in genes...The CRISPR/Cas9 com­plex is very efficient at introducing double stranded breaks (DSBs) into genomic DNA...generating gene knockouts, NHEJ repair does not allow introduction of specific sequence changes. To generate ...Accomplishing this usually involves simultaneously introducing a homologous DNA repair template, such as a sin­gle-stranded...in our lab. Our goal was to use the system to introduce disease-causing mutations in iPSCs (or correct... but had never been systematically analyzed: introducing silent CRISPR/Cas9 blocking mutations in either...target sequence once the desired edit has been introduced. We found that blocking mutations increased the...

...Callaway EM. Retrograde neuronal tracing with a deletion-mutant rabies virus. Retrograde neuronal tracing...a variety of cargoes, genetically engineered neurotropic viruses emerged as powerful tools for visualizing...that cell, and nascent RABV cross synapses to retrogradely infect neurons that project to the starter cell...occurs, subsequent cycles of viral replication and retrograde transmission occur, spreading the virus through...RABV G protein is necessary and sufficient for retrograde transmission (Etessami et al., 2000, Wickersham...2007). Thus, RABV transsynaptic spread can be controlled by selectively supplying the G protein in trans...released from the starter cell and transmits retrogradely to first order neurons, via entry mediated by...

...AAV9 serotypes have strong neuronal tropism, AAV5 targets both neurons and astrocytes, and AAV4 targets ...receptor subunits or neurotrophic factors. Another option uses optogentics to control neuronal activity,..., behavioral neuroscientists can now precisely control where and when certain genes are expressed to investigate...specific behavior. This blog post will give an introduction to behavioral neuroscience, and address experimental... in a laboratory, a high level of experimental control is required, because seemingly tiny external factors...Experimenter Interaction should be minimal and strictly controlled; optional handling animals Experiment Behavioral...channels that are chemically activated. Both are introduced in neurons and depending on the choice of channel...

...such as prototrophy, the ability to synthetize all compounds needed for growth, or auxotrophy, the inability...synthesize a compound needed to grow. Auxotrophy and prototrophy come into play during cloning if you’...lab created a yeast prototrophy kit containing 23 plasmids to complement auxotrophy. Negative selection...antibiotic resistance cassette and you aren’t introducing an additional antibiotic resistance cassette ...plasmid backbone contains your gene of interest. Auxotrophy Besides antibiotic selection, you can select ...you’ve designed your experiment using an auxotrophic strain of bacteria (one that doesn’t synthesize an ...amino acid), and a plasmid that complements the auxotrophy (i.e., synthesizes the critical amino acid). ...