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We narrowed to 63 results for: generation lentiviral packaging system

Showing: 31 - 60 of 63 results

  1. Viral Vectors 101: AAV Serotypes and Tissue Tropism

    Type
    Blog Post
    ...to target the central nervous system, including neurons, AAV5 is generally the best choice for targeting...Vectors 101: Systemic Capsids Resources on Addgene.org AAV Data Hub AAV Guide AAV Packaged On Request ...target specific tissues, even when administered systemically, without causing disease? Recombinant adeno-...good to be true! In a previous post, we covered systemic capsids, which allow AAVs to broadly spread throughout...difficult-to-target tissues like the central nervous system. In this post, we’ll dive into the opposite: AAV... and infect specific cells. This protein shell packages the vector’s genetic material, and variations ...the viral particle sneaks its way into cells. Generally, AAV serotypes have a primary cellular receptor...

  2. Retrovirus Plasmids

    Type
    Collection
    ... for more information about: γ-Retroviral packaging systems Frequently asked questions (FAQ) about retrovirus...γ-Retrovirus Envelope and Packaging Plasmids Common γ-retroviral envelope and packaging plasmids. ID Plasmid...12371 pCL-Eco Packaging Contains MoMLV gag, pol, and env Verma 35614 pBS-CMV-gagpol Packaging MoMLV gag and...retroviral plasmids at Addgene, including popular packaging, envelope, and transfer plasmids. Viral...Resources: γ-Retrovirus Guide Biosafety Guide Lentiviral Plasmids AAV Plasmids Viral Vectors 101 eBook...virus uses the reverse transcriptase in tow to generate a DNA provirus. This provirus then gets integrated...retroviruses called γ-retroviruses. γ-Retroviruses can package relatively high amounts of DNA (up to ∼8 kb), and...

  3. Adenovirus Plasmids

    Type
    Collection
    ...then be transfected into mammalian packaging cell lines to generate adenovirus particles. Read our Adenoviral...Additional Resources: Adenovirus Guide Virus Biosafety Lentiviral Plasmids AAV Plasmids Viral Vectors 101 eBook... adenoviruses in research include their large packaging capacity (>8 kb), high titers, and high levels...terms Adenoviral Strains Bacterial strains for generating adenovirus. ID Strain Description PI 16398 BJ5183...Adenoviral A block for the AdenoBuilder genome assembly system. Block 6 with deletion in E3 and insertion of GFP...Adenoviral A block for the AdenoBuilder genome assembly system; consolidates the regions present in pAd5-B6 ∆E3...Adenoviral A block for the AdenoBuilder genome assembly system. Block 1 with deletion in E1 and insertion of GFP...

  4. Hot Plasmids: Spring 2025

    Type
    Blog Post
    ...CMV HA-EGFP Puro (Addgene #236081), then generated lentiviral particles in HEK293T cells with pMD2.G (...tailored deactivation rates Addgene's own empty lentiviral backbones New recombinant antibodies for Glypicans...very own set of empty, HA-, and EGFP-expressing lentiviral backbones designed right here at Addgene. Here's...taken them for a test drive! We validated viral packaging and strong expression after inserting EGFP and...and provide step-by-step protocols for viral generation and stable cell line creation (Figure 2). Looking...launching later this year!  Find Addgene's own Lentiviral backbone plasmids here! Figure 2: Addgene...Addgene #12260). HeLa cells were infected with 96 h lentiviral supernatant and selected with puromycin. Puromycin-resistant...

  5. Choosing Your Perfect Empty Backbone

    Type
    Blog Post
    ...upgrade your basic mammalian expression system and go for a lentiviral or a retroviral backbone. This is considered...expression system and has its advantages; however, remember that you will need accessory packaging vectors...vectors to generate the virus. Bacterial vectors This system is great for overexpressing YGOI in an isolated...important thing you need to know is your expression system or environment. The host organism will determine...vivo (try pcDNA3-EGFP). If you can get away with generating transiently transfected cells, you will not need...

  6. CRISPR Library Amplification

    Type
    Protocol
    ...as these sequences are not efficiently packaged into lentiviral particles. See below for options if the...amplification. This protocol is designed to be as general as possible but note that individual libraries ...our protocol on Bacterial Transformation . The generalized pooled library amplification protocol is expected... a diagnostic digest and high-throughput next generation sequencing (NGS). Select a restriction enzyme...gel ( see restriction digest protocol here ). Lentiviral plasmids can recombine between their LTRs resulting...recombinant plasmid likely grow faster in the closed system of a crowded plate or culture. Try restricting ...

  7. A Look Back at One Year of Plasmid Sharing for COVID-19 Research

    Type
    Blog Post
    ...COVID-19 pandemic, the Krogan lab at UCSF generated lentiviral vectors expressing SARS-CoV-2 open reading...but one are cloned into pLVX vectors (for lentiviral packaging) and contain an IRES-Puro marker for stable...from Alejandro Balazs’s lab includes those for generating pseudovirus and those encoding different spike...expression plasmids available for many expression systems: bacterial, yeast, and mammalian. These plasmids...(2020) Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization...published a protocol and reagents for pseudotyping lentivirus with the SARS-CoV-2 spike protein. They deposited...

  8. Zhang Lab CRISPR Page

    Type
    Collection
    ...view: Zhang Lab General Cloning Protocol 237.2 KB SpCas9 Plasmids The CRISPR/Cas system can be implemented... formats: 1 vector system - lentiCRISPR - sgRNA and SpCas9 together 2 vector system - lentiCas9-Blast ...#61425 - #61427 are intended for lentivirus production : 61425 : lentiviral vector encoding dCAS9-VP64 with... P alindromic R epeats) is a microbial nuclease system involved in defense against invading phages and.... We have harnessed the type II CRISPR nuclease system to facilitate genome editing in mammalian cells...Nature Biotechnology 2013). We also have an older system using two cassettes to express the CRISPR RNA array... top GeCKO Screening Library GeCKO are pooled lentiviral CRISPR libraries (both human and mouse) for performing...

  9. Luciferase Plasmid Collection

    Type
    Collection
    ...plasmids are meant for expression in mammalian systems. ID Plasmid Luciferase Type(s) Description PI 64784...interest Scott Lowe 21375 pHIV-Luciferase Firefly Lentiviral vector for insertion of your gene of interest...N-terminal Nanoluc fusions using Gateway cloning. Lentiviral. Mikko Taipale 12563 PCD FL0X Firefly Insertion...Firefly Insertion of 5' promoter/enhancer regions. Lentiviral Nadav Ahituv 212935 pGL4.84(hRlucCP/Puro) RapidResponse...Renilla Insertion of 5' promoter/enhancer regions. Lentiviral Nadav Ahituv 108228 pGreen2x35SLUC Firefly, Renilla...21471 pLenti PGK V5-LUC Neo (w623-2) Firefly PGK Lentiviral expression of firefly luciferase Eric Campeau... pLenti CMV V5-LUC Blast (w567-1) Firefly CMV Lentiviral expression of firefly luciferase Eric Campeau...

  10. Viral Vectors 101: Types of viruses

    Type
    Blog Post
    ... not interchangeable between the two systems, other, more general, viral components such as a heterologous...-studied lentivirus is HIV, which many lentiviral tools have been derived from. Lentiviruses have slightly...larger packaging capacities than gamma-retrovirus, coming in around 9 kb. Unlike gamma, lentivirus can infect... 1982). To add insult to injury, retroviruses generally have moderate to high immunogenicity, meaning ...adenovirus have been identified so far! Adenovirus generally has high immunogenicity, but some strains have... expression of adenovirus cargo is high, it’s generally not as long lasting as AAV (up to several weeks... fact: All viruses discussed in this blog are generally broadly infective, with one exception. Blood cells...

  11. Addgene: The First Twenty Years

    Type
    Blog Post
    ... the Internet exploded. From day one, we built systems for now-common business practices, like accepting...Network Excellence Award for our electronic MTA system. We also started our blog and YouTube channel in... in 2013, which continue to grow today. “I generally refer people to Addgene for the basics of molecular...ready-to-use viral vectors from some of our most popular lentiviral and AAV vectors, as well as developing and sharing...expanding our viral vectors service to include AAV Packaged on Request! “I love Addgene. I think resources...

  12. CRISPR References and Information

    Type
    Collection
    ... vector system: lentiCRISPR v2 2 vector system: lentiCas9-Blast and lentiGuide-Puro packaging plasmids... Information Addgene Resources CRISPR Software General Resources CRISPR Protocols Addgene CRISPR Resources...CRISPOR batch (Link opens in a new window) for lentiviral screens. sgRNA Scorer 2.0 (Link opens in a new...species. Developed by the Kate O'Connor-Giles Lab . General Resources CasPEDIA (Link opens in a new window)...window) An encyclopedia of Class 2 CRISPR systems with wiki entries describing enzyme activity, experimental...Link opens in a new window) CRISPR/Cas9 reagent generation; gRNA design; HDR design; Deep sequencing Cas9...PDF, 255 KB Zhang sgRNA cloning lentiCRISPR v2 packaging plasmids: pVSVg , psPAX2 ; positive control: CMV-EGFP...

  13. 27 Hot Plasmids from 2016

    Type
    Blog Post
    ...the authors packaged it into lentivirus and used the resulting lentiviral library to generate a population... CRISPR Library. This complex second-generation CRISPR lentiviral library targets nearly all human protein-coding... cell systems and diverse model organisms. Addgene’s newest TALEN kit, the FusX assembly system from Stephen...obtained from the CDC and cloned into a third generation lentiviral transfer vector. Each gene has been FLAG-tagged...how these systems work. Researchers screening protein-protein interactions in mammalian systems have many...protein-protein interactions in mammalian systems. The DULIP system is comprised of Gateway-compatible plasmids...somatic hypermutation to generate diverse antibodies; in the CRISPR-X system, AID*Δ similarly produces...

  14. CRISPR Guide

    Type
    Collection
    ...will be generated close enough to cause a DSB. The nickase system is utilized in many CRISPR systems to achieve...using dual nickases to generate a knockout or gene edit; or using Cas9 to generate large genomic deletions...Cas9 (CRISPR Type II) systems, while AcrV inhibits Cas12a (CRISPR Type V) systems. There are rare cases...The two main classes of CAST systems are type I and type V-K. Type I systems use a type I Cas effector, ...), while type V-K systems use a type V-K Cas effector, Cas12k. No matter the system, the Cas effectors...activity. Each system also includes Tn7-like transposase components. Type I (generally) includes tnsA ...CRISPR libraries What Are Pooled Lentiviral CRISPR Libraries? Pooled lentiviral CRISPR libraries are a heterogeneous...

  15. Viral Vectors 101: Transductions

    Type
    Blog Post
    ...viral packaging elements that will ensure the virus gets produced, and viral particles are packaged, when...Resources Resources on Addgene.org Generating Stable Cell Lines with Lentivirus Virus Handling and Storage Addgene...is then typically transfected along with viral packaging plasmids into a viral production cell line, usually...cell line, viral particles will be produced and packaged, resulting in functional virions. These viral ... stably integrate (unless you used a transposon system, CRISPR targeted strategy, etc.) and episomal expression...perform the transduction with: either retrovirus, lentivirus, adenovirus, or AAV. Then, your desired DNA to... some viruses integrate into the host genome (lentivirus and retrovirus) while others are non-integrating...

  16. Viral Vectors 101: Biosensors

    Type
    Blog Post
    ...pretty simply – they are systems that measure biological phenomena by generating an output which is directly... to generate a quantitative signal upon recognizing the analyte. This signal depends on the system and...transducer will generate a numerical value which represents the amount of analyte present in the system at a given...choose lentivirus, gamma retrovirus, or AAV as your vehicle, we’ve got you covered with packaging plasmids...biosensors and how to use viruses to set up these systems. Components of a biosensor Some of the biosensors...the amount of a given factor within a biological system. Basically, biosensors provide quantifiable readouts...that you care about within your cellular or animal system. This ‘thing’ could be a neurotransmitter, such...

  17. Viral Vectors 101: An Introduction to AAV

    Type
    Blog Post
    ... is a single stranded, Parvoviridae DNA virus, packaged in a non-enveloped icosahedral capsid, that can...to a number of different cell types even when systemically administered in an organism. These features ...the type of capsid (outer protein shell) used to package an rAAV vector; these capsids allow the virus to... over 4.7 kb. Co-infection rates for AAVs are generally high enough for this approach to work, although...design single rAAV vectors with multiple genes packaged in them and use elements such as IRES or 2A to...rescued from the vector plasmid and individually packaged into the AAV capsids. As a genome conversion in...see if others have used relevant viruses for your system. This post was contributed by guest blogger Didem...

  18. Viral Vectors 101: Producing Your rAAV

    Type
    Blog Post
    ...replication. The most common production system, however, is a “helper-free” system, in which the required adenoviral...titer rAAV (including Addgene!), but you can also generate rAAVs in your own lab with standard molecular ...adenoviral helper genes are supplied on two different packaging plasmids: the pHelper plasmid containing Ad E2A...serotype-specific capsid genes.  In this helper-free system, HEK293T cells, expressing Ad E1A and E1B, can ...our ddPCR for AAV quantitation blog post.  We generally do not recommend using ELISA-based titering assays... of AAV-2 particles via a novel capsid ELISA: Packaging of genomes can limit production of recombinant...resources on the Addgene blog Parts of the AAV Packaging PlasmidParts of the AAV Transfer PlasmidAAV Variables...

  19. Viral Vectors 101: Virus Safety

    Type
    Blog Post
    ...? That’s okay! In this article we will discuss general viral safety considerations and best practices ...infectivity in check. Engineering controls, safety systems that are built into how something functions, were...virus into different plasmids. For a retroviral system this looks like the genetic cargo in one plasmid...Gag structural protein on yet a third plasmid (packaging plasmid). Viral production then proceeds by transfection....   Figure 1: Separation of retroviral packaging components onto distinct plasmids for safety.  ... and the same holds true for types of viruses generated in the lab. The four most common types of viruses... lab and require additional safety measures.   General viral handling safety   Containment and personal...

  20. AAV Vector Quality Control: Going the Extra Mile with NGS

    Type
    Blog Post
    ...sequence all the DNA packaged inside the viral particles (Figure 1). Briefly, packaged DNA is extracted from...more rigorous quality control, including next generation sequencing (NGS) of the viral genome to confirm... the helper plasmids or the cell genome can be packaged inside the vectors during production. These impurities... the virus itself. While these impurities are generally considered innocuous for research-grade vectors... to determine the identity and serotype of the packaged DNA and look for potential contaminants. The ...mentioned earlier, it is common to find DNA from the packaging cell genome, bacterial genomes, cloning vectors...the recombinase for transgene expression. Once packaged in AAV, this pre-recombined DNA could lead to ...

  21. DAP arrays for multiplex gene editing

    Type
    Blog Post
    ... Lab packaged MBE and MPE elements for deliveries via adeno-associated virus (AAV) and lentivirus, demonstrating...and prime editing. How do you then set up an MBE system that contains a double-win for both efficiency ...to develop a strategy that would independently generate multiple gRNAs for nCas9-BE, or any other kinds...strategic advantages in gene editing, while keeping the system to minimal size. To do so, they harnessed the ...promoters, has limited the number of gRNAs that can be packaged into viral vectors for potential in vivo applications...drive-and-process.” To optimize DAP use, they developed a generalizable method to acquire the most effective DAP array...

  22. AAVs in Retinal Gene Therapy

    Type
    Blog Post
    ...integration into the genome, and its large DNA packaging capacity is well suited for larger transgenes....humans, and its low immunogenicity is critical when systemic delivery of high doses of vectors are needed. ...this gene can be targeted to RPE cells using a lentiviral vector and a clinical trial is ongoing. Moving...vectors (AAVs) to treat an inherited retinal degenerative disease called Retinitis Pigmentosa. I quickly...other viral vectors such as adenovirus (AdV) and lentivirus (LV) are known to transduce retinal cells, AAV...pigmentosa, achromatopsia, and age-related macular degeneration are ongoing and some will hopefully follow in...muscular dystrophy, spinal muscular atrophy, neurodegenerative diseases and metabolic diseases are also underway...

  23. Viral Vectors

    Type
    Collection
    ...and generate virus (right), similar to how natural viruses can be delivered to a host and generate virus... Nolan Lab Retroviral Systems Trono Lab Lentivector Toolbox Guide to Generating Recombinant Adenoviruses...can direct long-term transgene expression, but generally are not permanently integrated into the host genome... genome. AAVs are limited by their small packaging capacity. Adenovirus Plasmids | Guide Adenoviruses ...an organism. Figure 1. Using viral plasmids to generate virus. A) Viral plasmids have been engineered ...genetic material (termed the viral genome) that, in general, encodes for more viruses. When a virus infects...researchers can introduce plasmids to a host to generate virus. Furthermore, these plasmids can be modified...

  24. Important Considerations When Using AAVs

    Type
    Blog Post
    ...cells of interest. Although they have a small packaging capacity, AAV vectors were suitable for our needs...(such as AAV2) (4). Otherwise, using a specific system for controlling gene expression such as Cre-lox... eye, skeletal muscles, and the central nervous system. Currently, AAVs are being used to deliver CRISPR...mediated gene expression and tropism in mice after systemic injection." Molecular Therapy 16.6 (2008): 1073...Grieger, Joshua C., and Richard J. Samulski. "Packaging capacity of adeno-associated virus serotypes: ...Self-complementary recombinant adeno-associated viral vectors: packaging capacity and the role of rep proteins in vector.... 11. Wouterlood, Floris G., et al. "A fourth generation of neuroanatomical tracing techniques: exploiting...

  25. Plasmids 101: Terminators and PolyA signals

    Type
    Blog Post
    ...signals in combination with certain viral packaging systems has been associated with reduced viral titer...to express genes in a given host cell type are generally broken down into two broad categories, prokaryotic...Plasmid DNA in both prokaryotic and eukaryotic systems must be transcribed into RNA, which occurs in three...polyadenylation vary across cell types, but polyadenylation generally serves to promote transcript longevity in eukaryotes...Prokaryotic termination mechanisms fall under two general categories: rho-dependent and rho-independent. ...not usually employed in plasmid-based expression systems, so these will not be detailed here, but additional...termination DNA transcription Polyadenylation: General overview BGH rbGlob SV40 Resources on the Addgene...

  26. Viral Vectors 101: Viral Applications

    Type
    Blog Post
    ...simultaneously in your system instead of just one at a time. Many of these libraries are packaged and delivered...before building one from scratch! Why deliver these systems virally instead of by another method? Viral delivery...integration of a plasmid. Viral transduction is also generally less toxic to cells than transfections, preserving...traditional transfection method. This is because viral systems have a high infection frequency and the viral titer... targeting features make them an ideal delivery system for human patients. Multiple viral-based gene therapies...is more than one type of virus to choose from (lentivirus, retrovirus, AAV, etc.). When you’ve decided ...

  27. Lentiviral Vector Guide

    Type
    Guide
    ... different generations. Lentiviral Generations First-generation First-generation lentiviral plasmids are...second-generation lentiviral transfer plasmids must be used with a second-generation packaging system because...Third-generation systems are considered safer than second-generation systems because the packaging components...Read our lentiviral guide to learn about lentiviral components, generations, lentiviral production, and... Guides Lentiviral Vector Guide Lentiviral Vector Guide Jump To: Lentiviral Vectors Lentiviral Generations...Third-generation transfer plasmids can be packaged by either second- or third-generation packaging plasmids...and third-generation systems, please refer to Table 1. Figure 4: Third-generation lentiviral plasmids....

  28. Plasmids 101: Using Transposons in the Lab

    Type
    Blog Post
    ... when packaging viral vectors.   Let’s take a closer look at three different transposon systems adapted...cholerae that encodes a Type I-F CRISPR-Cas system. This system has four major components: 1) a CRISPR RNA...We often think of DNA as inert. It generally stays put, which makes it easy to locate in a genome. But...eukaryotic genomes.  Components of a transposon system Transposon: The transposon is the DNA sequence...is ~100-fold more efficient compared to first generation SB transposase (Mátés et al., 2009). hySB100x...ve learned about some of the popular transposon systems, let’s take a look at how they can be used in the...transposon insertion sites are detected with next generation sequencing, and analyzed to identify which insertions...

  29. Gamma-Retroviral Vector Guide

    Type
    Guide
    ...lentiviral packaging systems, and vice versa. Gamma-retroviral vectors do not have defined “generations” of ...flanked by LTRs; ~8 kb packaging capacity Packaging plasmid — contains packaging genes gag and pol Envelope...similar to lentiviral packaging methods . The three plasmids described above (envelope, packaging, and transfer...plasmids, as lentiviral vectors do. For more about lentiviruses, see Addgene's lentiviral vector guide...of these packaging components. Therefore, gamma-retroviruses may not be efficiently packaged by lentiviral...are co-transfected into the HEK293T packaging cell line. This system provides the greatest flexibility ... . Production Using Packaging Cell Lines Gamma-retroviral vectors can be packaged using helper-free cell...

  30. Plan Your Experiment

    Type
    Guide
    ...want to: Generate complete and permanent loss of gene expression or function (knockout)? Generate a specific...Many different activator systems exist, including the multi-plasmid SAM system Activate Design and Clone...the optimal system will likely depend on your cell type. Figure 2: Different delivery systems utilized for...summarizes ways that the CRISPR/Cas system can be delivered to cells. The system may be delivered using viral...production, see our viral vector guides . Lentiviral vectors Lentiviral vectors are a popular option for producing...vectors is that the packaging limit (how large of a DNA sequence you can actually package into your viral ... vector) is smaller than that of lentivirus. Thus, AAVs are generally only compatible with smaller Cas...
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