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We narrowed to 65 results for: lentivirus expression plasmid

Showing: 21 - 40 of 65 results

  1. New Tools Enable CRISPRa for Neuroscience Applications

    Type
    Blog Post
    Published
    July 2, 2019, 12:51 p.m.
    ... Dual lentivirus system with ubiquitous expression of sgRNA (top) and neuron-specific expression of dCas9... for generating the SPH mouse. (B) AAV plasmids used to express CRE under neuron-specific promoters (aCaMKII... (i.e., how many sgRNA expression cassettes will fit). Cre-dependent expression of the SPH activator paired...neuronal populations.  The Yang Lab’s AAV plasmids used to express Cre under neuron-specific promoters, including...Savell et al., 2019). This dual lentivirus system pairs ubiquitous expression of sgRNA(s) and mCherry (vector...without inducing expression of the non-targeted variant. Moreover, isoform-specific expression of Bdnf resulted...comprehensive CRISPR guide Find CRISPR plasmids and resources Find CRISPRa plasmids ...

  2. Making CRISPR Plasmids Using Fragmid

    Type
    Blog Post
    Published
    Aug. 29, 2024, 1:15 p.m.
    ...restriction digest, and then plasmid sequencing if you can. To quote Doench, “Whole plasmid sequencing is just ...correctly banded clones for plasmid sequencing and found 98% of the plasmids were perfectly correct. Because...Mammalian Lentivirus pRDA_512 (RSV-driven; lentiGuide-based) Mammalian Lentivirus pRDA_722...pRDA_757 (Piggybac; constitutive expression) Mammalian Plasmid pRDA_791 (contains only orio and...Mammalian or drosophila) Delivery type (lentivirus, AAV, transposon, Plasmid) Destination vector (dropdown options...the assembled plasmid. It shows you each component, with the ID of the component plasmid, component name...the component plasmids on the Addgene website. Step 4: Naming your assembled plasmid and generating...

  3. Viral Production at Addgene

    Type
    Blog Post
    Published
    Oct. 24, 2023, 1:15 p.m.
    ...the transfer plasmid, a plasmid encoding rep and serotype-specific cap genes, and a plasmid encoding adenoviral.... coli. In addition, plasmids are typically prepared using endotoxin-free plasmid purification kits.   ...against unique regions of the transfer plasmid.   Lentivirus resources Browse lentiviral preps available...contamination is minimized by using an endotoxin-free plasmid purification protocol. To minimize the immunogenic...vectors are tested in vitro and in vivo for gene expression and/or function. These data are sometimes reported...mL, or both. Two days later, Cre-dependent GFP expression was detected with direct fluorescence. GFP was...was not detected in the absence of Cre. mCherry expression alone was detected. pAAV-Ef1a-mCherry-IRES-Cre...

  4. Viral Vectors 101: Virus Safety

    Type
    Blog Post
    Published
    June 22, 2023, 1:15 p.m.
    ...transfer plasmid), the envelope (Env) gene on a separate plasmid (envelope plasmid), and the polymerase ...different plasmids. For a retroviral system this looks like the genetic cargo in one plasmid (the transfer...Gag structural protein on yet a third plasmid (packaging plasmid). Viral production then proceeds by transfection... transfection of all three plasmids into a producer cell line, such as HEK-293T, production of viral particles... and Pol genes are not encoded in the transfer plasmid, the component that physically integrates into ... retroviral packaging components onto distinct plasmids for safety.    Viral cargo-specific safety...viruses grown in the lab are gamma retrovirus, lentivirus, adenovirus, and adeno-associated virus (AAV)...

  5. Hot Plasmids - December 2019 - CRISPR pooled library, LipoGlo, CRISPRa, and Voltage Imaging Tools

    Type
    Blog Post
    Published
    Dec. 10, 2019, 1:58 p.m.
    ...highlight a subset of the new plasmids in the repository through our hot plasmids articles. These articles ...recent plasmid deposits and we hope they'll make it easier for you to find and use the plasmids you need...need. If you'd ever like to write about a recent plasmid deposit please sign up here.        A retroviral...However, some cell types are poorly transduced by lentivirus, and thus screening with available CRISPR libraries...Primary murine T cells cannot be transduced by lentivirus and have been difficult to manipulate genetically...type 2 (Th2) cells from mice that constitutively express Cas9 and screen for genes that affect Th2 differentiation...differentiation and activation, using expression of differentiation factors as the screen readout. This retroviral...

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

    Type
    Blog Post
    Published
    March 7, 2017, 3:30 p.m.
    ...cells to express Cas9. In these particular experiments, we generated A549 cells stably expressing Cas9 via...pools of A549 cells transduced with Cas9 expressing lentivirus at the indicated multiplicity of infection...wildly variable Cas9 expression (Figure 1). This is a problem because Cas9 expression affects growth rate...variable Cas9-expression was across the different cells (Figure 3).   Figure 2: Cas9 expression in monoclonal...Checking Cas9 expression Overview: By Western blotting (Figure 2, pool), I knew the cells expressed Cas9, but...thought I could see GFP expression with a fluorescence microscope, but the GFP expression was so low (even in...against GFP expression, but I didn’t. 2. In the original publication they see GFP expression in about 20%...

  7. Delivery Methods for Generating iPSCs

    Type
    Blog Post
    Published
    April 17, 2018, 1:37 p.m.
    ... as a donor plasmid containing the transposon, along with a helper plasmid that expresses the transposase...lingering expression of reprogramming factors. Non-replicating Plasmids: Generating iPSCs with plasmid-based...plasmid-based expression requires serial transfection of 1 or 2 plasmids that express the reprogramming factors...mutagenesis, transgene reactivation, residual expression Lentivirus Integrating High Low Efficient and Stable...reprogramming. Additionally, lingering expression or re-expression of viral transgenes in iPSC-derived cells...cassette that expresses multiple reprogramming factors driven by one promoter. While lentivirus has similar... generating iPSCs by plasmid transfection where 2 of 11 clones tested had plasmid integration. Another...

  8. What's New in CRISPR - December 2019

    Type
    Blog Post
    Published
    Dec. 17, 2019, 2:37 p.m.
    ...CRISPR plasmids available at Addgene. We will still periodically focus on specific CRISPR plasmid tools...CRISPR guide Find CRISPR plasmids by function Find recently deposited plasmids ...of bacteria that can cause disease in humans. Expression of this system is tunable via a rhamnose-inducible...lentiviral capsid-based bionanoparticle Transient expression of CRISPR/Cas9 in cells has advantages like reduced...packaging Cas9/gRNA ribonucleoproteins for transient expression. They replaced the tetraloop of the sgRNA scaffold... aptamer binding protein for packaging into a lentivirus-like particle. Read the paper in Nucleic Acids...Acids Research Find the gRNA and packaging plasmids   Cytosine deaminase for RNA editing Feng Zhang’s ...

  9. Tips for a 1st time AAV user (by a Rookie AAV user)

    Type
    Blog Post
    Published
    Oct. 23, 2018, 11:49 a.m.
    ...packaging plasmids. The cells need three different plasmids to produce AAV: 1) the RepCap plasmid which provides...AAV plasmid standard and AAV transfer plasmids. It’s easy to contaminate a workspace with plasmid, not...protein shell, or capsid; 2) the pHelper plasmid which expresses adenovirus genes which help mediate AAV...going to lie, AAV transfer plasmids are kind of a pain to work with. These plasmids contain ITRs which are...AAV viral replication; and 3) the transfer plasmid which encodes a transgene of interest that’s packaged...structures that are prone to deletion from the transfer plasmid. The good news is there’s a few simple ways to ...multiple bacterial cultures of your AAV transfer plasmid at 30 °C instead of 37 °C and then screen for ITR...

  10. CRISPR 101: Epigenetics and Editing the Epigenome

    Type
    Blog Post
    Published
    June 24, 2020, 5:45 p.m.
    ...from Rudolf Jaenisch’s lab in plasmid form or as ready-to-use lentivirus. Figure 2: dCas9-Tet1 ... are an additional layer of control over gene expression that go beyond genomic sequence. Dysregulation...modifications are correlated with alterations in gene expression, but causality and mechanisms remain unclear....that help package DNA, were associated with gene expression or silencing. To alter DNA modifications, researchers...Charles Gersbach’s lab has deposited mammalian expression constructs including pcDNA-dCas9-p300 Core and...well as pLV-dCas9-p300-P2A-PuroR for lentiviral expression. Figure 1: dCas9-p300 adds H3K27ac marks...cytosine demethylation in mammalian cells. This plasmid is used with pcDNA3.1-MS2-Tet1-CD to decrease methylation...

  11. Viral Vectors 101: The Retroviral Lifecycle

    Type
    Blog Post
    Published
    July 27, 2023, 1:15 p.m.
    ... the lentiviruses (like HIV-1) — that have been developed into lentiviral vectors. Lentiviruses are sometimes...instead get expression of the sensor, marker, or whatever other tool you chose to express. In the case...that the virus must have its structural proteins expressed from a reverse-transcribed piece of DNA called...is done, and the transgenic genome will simply express from whatever promoters were included in the vector...And there you have it, the full “life cycle” of expression of selfish, lazy genes of a non-living viral ...Addgene's Lentiviral Guide Most popular lentiviral plasmids γ-Retrovirus Guide References Cullen, B. R. (...

  12. 15 Years of Addgene: The Top 15 Plasmids

    Type
    Blog Post
    Published
    Jan. 8, 2019, 1:54 p.m.
    ...vectors and an envelope expressing plasmid, such as our second most requested plasmid of all time, pMD2.G.... the envelope expressing plasmid pMD2.G. Find pMDLg/pRPE. scramble shRNA - This plasmid from David Sabatini...(PX461) expresses both a Cas9 protein and a gRNA. But instead of Cas9, this plasmid expresses Cas9n (a...generation lentiviral packaging plasmid from the Didier Trono lab that expresses the HIV gag, pol, rev, and...Didier Trono lab is pMD2.G, a VSV-G envelope expressing plasmid. Envelope proteins determine which types ...wide range of applications. This plasmid was the top requested plasmid of 2017. Find pMD2.G. pSpCas9(BB...BB)-2A-GFP (PX458) - This plasmid from the Zhang lab expresses Cas9 from Streptococcus pyogenes with an...

  13. Plasmids 101: The protein expression toolbox

    Type
    Blog Post
    Published
    Feb. 16, 2023, 7:05 p.m.
    ...permanently induce expression? Check out our Retro expression vector and Lenti expression vector. Turn it...ablate protein expression. If you need the ability to control when you turn off expression for good, the...inducible expression (more on this below) will both achieve temporary repression of protein expression. Addgene... a gene (cDNA) can be done by either plasmid or virus. A plasmid carrying your cDNA can be targeted for...appropriate promoter to express your cDNA, depending on the level of expression you would like. Check out...choosing. Viral expression is another mechanism to integrate your choice cDNA. Both lentivirus and retrovirus.... Whether you want to express or repress, good luck on your protein expression journey! Resources and...

  14. Single-cell tracking of lineage and identity with CellTag

    Type
    Blog Post
    Published
    May 9, 2019, 1:15 p.m.
    ...cell page Find plasmids for reprogramming and differentiation Check out our lentivirus guide Sign up to...), now allows scientists to identify the gene expression patterns unique to the few cells that successfully...Unfortunately scRNA-seq only captures the gene expression of cells at a single time point, making it difficult...difficult to investigate how early expression patterns influence reprogramming outcomes. CellTag from ...single-cell level, thus linking ancestral gene expression patterns with reprogramming outcomes (Biddy et...CellTags were detected over a 10-week period. Their expression decreased over time but they were not silenced...MEFs were reprogrammed into iEPs by retroviral expression of transcription factors Foxa1 and Hnf4α, a process...

  15. X-CHIME: Context Dependent Germline Knockout in Immune Cells

    Type
    Blog Post
    Published
    Jan. 16, 2024, 6:13 p.m.
    ...and lentiviral expression vectors (Table 1). The four systems Name Function Plasmid Transduction...deposit also includes pXPR_071, a plasmid that leads to higher titer lentivirus preparations than the first ...2019b). CHIME works through transduction of Cas9-expressing (Rosa26-Cas9) hematopoietic stem cells with a...first generation CHIME (pXPR_053). Find X-CHIME plasmids here!   Marty LaFleur is a Postdoctoral Fellow in...

  16. Multiplexed Overexpression of Regulatory Factors (MORF) collection

    Type
    Blog Post
    Published
    March 30, 2023, 1:15 p.m.
    ...Addgene.org Plasmid Pooled Libraries Pooled library amplification Zhang Lab CRISPR Plasmids The Human TFome...factors (TFs) act as the gate keepers for gene expression, turning transcription on and off by binding ...binding proximal to their target genes. Since gene expression patterns determine everything from stem cell differentiation...(aka the MORF Collection) and an accompanying expression atlas, including over 3,500 human TF isoforms...TF library, with each cell receiving only 1 TF expression construct. Cells were cultured following a differentiation...from Joung et al.)    This atlas linked TF expression to differentiation outcomes as well as global...reference cell types generated by the human fetal expression atlas. Computational analysis was used to infer...

  17. 27 Hot Plasmids from 2016

    Type
    Blog Post
    Published
    Dec. 22, 2016, 3:03 p.m.
    ...transfer vector for in-vivo expression and plasmids optimized for dCas9-VPR expression in drosophila and yeast...Cas9 nickase, and Cas9 activator expressing plasmids that can co-express 1-8 gRNAs. Ordon et al., 2016 tested...Neurosci 2016. PubMed PMID: 26950006 Plasmid ID Plasmid Name Plasmid Type 74309 pcDNA3.0-TRPV4-p2A-ferritin-p2A-mCherry...highlight a subset of the new plasmids in the repository through our hot plasmids articles. These brief articles...features and applications of a partiular plasmid or set of plasmids. We hope that these articles make it ... you to find and use the plasmids you need. You can find all the hot plasmids from 2016 below. With over...over 45,000 plasmids, we can't write posts for every great plasmid that comes into the repository, but...

  18. Important Considerations When Using AAVs

    Type
    Blog Post
    Published
    June 13, 2017, 2:30 p.m.
    ... have found that expression occurs quickly and results in long and stable expression (up to 9 months in...the onset of gene expression. However, it has yet to be determined what gene expression looks like at 3 ...facilities (Biosafety Level-1). Other possible tools, lentiviruses for example, are not approved for use in vivo...Response: Compared to other viral vectors (e.g. Lentivirus, Adenovirus) AAVs cause the least amount of immune...AAV2 can transduce many tissue types, it has low expression in several brain regions where other serotypes... under study. Many AAVs will show significant expression in a variety of cell types, but you should try...transport desirable or not desirable? If diffuse gene expression is tolerated, then axonal transport is fine and...

  19. Plasmids 101: Terminators and PolyA signals

    Type
    Blog Post
    Published
    March 31, 2016, 2:30 p.m.
    ...Plasmids designed to express genes in a given host cell type are generally broken down into two broad...Download our Plasmids 101 eBook to get all the background you need to start working with plasmids! What are...terminators are not usually employed in plasmid-based expression systems, so these will not be detailed...at the end.  Nearly all common bacterial expression plasmids use Rho-independent terminators, which include...termination with polyadenlyation. Mammalian expression plasmids are primarily used to create mRNA and the...Polyadenylation Signal Substantially Increases Expression Levels of Lentivirus-Delivered Transgenes but Has the Potential...Promoter Controls Gene Expression Learn about New Cloning Techniques Read all of our Plasmids 101 Posts Resources...

  20. CRISPR 101: Mammalian Expression Systems and Delivery Methods

    Type
    Blog Post
    Published
    Sept. 24, 2015, 2:30 p.m.
    ...donor plasmid or single-stranded DNA oligonucleotide, so you will need to make sure your expression system...included with each expression system as well as suitable applications. Expression System Components ...System Application Mammalian expression vector Promoter driving Cas9 expression can be constitutive or inducible...or electroporation. Transient expression of CRISPR components, expression decreases as RNA is degraded ...using cationic lipids. Transient expression of CRISPR components, expression decreases as gRNA and Cas9 protein... provide an overview of some CRISPR mammalian expression systems, the typical applications for each, and...gene? Do you want to activate or repress gene expression? Are you trying to create a single point mutation...
Showing: 21 - 40 of 65 results