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We narrowed to 921 results for: tro

Showing: 161 - 180 of 921 results

  1. Delivery Methods for Generating iPSCs

    Type
    Blog Post
    ...Moloney murine leukemia virus (MMLV)-derived retroviruses. Retroviral vectors, such as pMXs or pMSCV, infect...delivery methods. Retrovirus has a cloning capacity of ~8 kb, so either a single polycistronic vector or multiple...10 kb) than other retroviral vectors. This makes it possible to use a polycistronic cassette that expresses...Mechanism Efficiency Safety Pros Cons MMLV-derived Retrovirus Integrating High Low Efficient and Stable Genome...Need to sequence cells to verify excision didn’t introduce mutations Adenovirus Non-integrating Low Medium...required for translational use. Viral Vectors Retroviral: Some of the first iPSCs were generated with ... corresponding endogenous pluripotency genes. Retroviral vectors are prone to incomplete silencing of ...

  2. Plasmids 101: Stringent Regulation of Replication

    Type
    Blog Post
    ...DNA.  Plasmids are said to be under stringent control when they depend on the host cell’s initiation ...replication independently are said to be under relaxed control, as they only require the host's replication machinery...replication mechanism of plasmids under stringent control, such as pSC101. DNA replication and regulation...round of replication. Initiation is the primary control point of DNA replication in bacteria, so the process... the bacterial chromosome and for stringently controlled plasmids. Binding of IHF (green) redistributes...binding is necessary to initiate replication, controlling the amount of active DnaA available is one of...source, ATP binding is one more way for the cell to control DNA replication. A variety of other proteins also...

  3. Plasmids 101: The Promoter Region – Let's Go!

    Type
    Blog Post
    ...target genes. The sequence of the promoter region controls the binding of the RNA polymerase and transcription...promoters is commonly employed in lentiviral and retroviral constructs and we will discuss these in a future... all the time while others are more carefully controlled. Regulated promoters might act only in certain... light. In the cell, promoters themselves are controlled by still other regulatory factors: enhancers,...Additional considerations CMV General expression mRNA Strong mammalian expression promoter from the human cytomegalovirus...some cell types. EF1a General expression mRNA Strong mammalian expression from human elongation factor...promoter hybrids. CAG General expression mRNA Strong hybrid mammalian promoter Constitutive  Contains...

  4. 27 Hot Plasmids from 2016

    Type
    Blog Post
    ... under the control of the cI857 temperature-sensitive repressor. The temperature-controlled expression...activity in the Co-IP compared of a control Co-IP indicates stronger interaction. The system has been demonstrated...DECEMBER 2016 Restoring yeast prototrophy with plasmids Though auxotrophic markers are used frequently ...complement strains with multiple auxotrophies or using different auxotrophic backgrounds can lead to disparate...their Yeast Prototrophy Kit, a set of plasmids designed to complement unused auxotrophies in Saccharomyces... at a dose and duration sufficient to kill all control cells and surviving “mutant” colonies were deep...GTPases involved in cell signaling pathways that control many cellular processes such as differentiation...

  5. Viral Vectors 101: Viral Vector Elements

    Type
    Blog Post
    .../gamma-retrovirus Lentiviruses are a class of retrovirus (so all lentiviruses are retroviruses, but not...not all retroviruses are lentiviruses.) This means that lentivirus and gamma-retrovirus share a lot of...as tools in the lab. We will discuss the gamma-retrovirus, lentivirus, adenovirus, and adeno-associated...somatitis virus G glycoprotein (VSVG). VSVG is a broad tropism envelope protein that can also be pseudotyped to...the common components outlined above for gamma-retroviruses. While not required for lentiviral production...   Fig. 1 - Viral vector components of retrovirus  Adeno-associated virus AAV’s elements are...are quite different from gamma-retrovirus, but they perform very similar functions to infiltrate the host...

  6. Viral Vectors 101: AAV Variables That Matter

    Type
    Blog Post
    ...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...

  7. Site Directed Mutagenesis by PCR

    Type
    Blog Post
    ... 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...

  8. 28 Hot Plasmid Technologies from 2015

    Type
    Blog Post
    ...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...

  9. Viral Vectors 101: Viruses as Biological Tools

    Type
    Blog Post
    ...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...

  10. Viral Vectors 101: Pseudotyping

    Type
    Blog Post
    ...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...

  11. Tips for a 1st Time CRISPR User (by a 1st Time CRISPR User)

    Type
    Blog Post
    ...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...

  12. Opto-Nanobodies: Using Light to Manipulate Cell Signaling and Protein Purification

    Type
    Blog Post
    ... 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...

  13. Plasmids 101: Yeast Vectors

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    Blog Post
    ... 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...

  14. Antibodies 101: The Basics of Western Blotting

    Type
    Blog Post
    ...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...

  15. Hot Plasmids Spring 2024

    Type
    Blog Post
    ...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...

  16. Viral Production at Addgene

    Type
    Blog Post
    ...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...

  17. Hot Plasmids April 2018 - Protein Degradation, Nanoscopy, FIRE-Cas9, and Yeast Expression Tools

    Type
    Blog Post
    ...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...

  18. Hot Plasmids - March 2019 - Anti-CRISPR, 2in1 Cloning, Fluorescent Voltage Indicators, and Photoswitchable Proteins

    Type
    Blog Post
    ... 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 ...

  19. Viral Vectors 101: Optogenetic Tools

    Type
    Blog Post
    ...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...

  20. Your Lentiviral Plasmid FAQs Answered

    Type
    Blog Post
    ...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...
Showing: 161 - 180 of 921 results