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We narrowed to 54 results for: generation lentiviral packaging

Showing: 21 - 40 of 54 results

  1. Viral Production at Addgene

    Type
    Blog Post
    Published
    Oct. 24, 2023, 1:15 p.m.
    ...Concentrated lentiviral preparations (“-LVC” catalog items) are generated from lentiviral preparations...produce both our AAV preps and our lentiviral preps.   Viruses are generated using standard methods that have...produced in 293T cells using the 2nd generation psPAX2 and pMD2.G packaging system developed by the Trono lab...being frozen. Specifically, lentiviral particles are collected from the lentiviral preparation by precipitation...for in our reported titers. Lentiviral vectors are titered using a lentiviral droplet digital PCR (ddPCR...Browse our AAV vector collection. Lentivirus Production Lentiviral preparations ("-LV” catalog items)...of the transfer plasmid.   Lentivirus resources Browse lentiviral preps available from Addgene. After...

  2. Top Requested Lentivirus and AAV of 2016

    Type
    Blog Post
    Published
    Jan. 6, 2017, 3:56 p.m.
    ...inventory to 25 lentiviruses and 25 AAVs. These viruses have been distributed in over 200 packages to more than...provide protocols and useful tips for generating your own lentivirus. Secondly, you can use a LentiCas9-...typically cytotoxic, which makes generation of high titer Cas9 lentiviruses challenging. This cytotoxicity...lentiCas9-Blast Most of Addgene’s lentiviral inventory is based on CRISPR tools, though we do provide a... plasmid. LentiCas9-blast virus can be used to generate Cas9-expressing cell lines, which can then be ...rate variability when using lentiCas9-blast to generate Cas9-expressing cells. To ensure that this growth...confound your experimental data, we recommend generating a monoclonal Cas9-expression line prior to performing...

  3. Hot Plasmids Spring 2024

    Type
    Blog Post
    Published
    May 23, 2024, 1:15 p.m.
    ...within the lentiviral 3’ long terminal repeats (LTR). The 3’ LTR is duplicated during lentiviral integration...license. In this iteration, the size of the lentiviral insert was minimized to reduce recombination ...III transcript, the sgRNAs were encoded on the lentiviral minus strand in the 3’ LTR. These optimizations...proteins and a critical component for viral genome packaging. Addgene recommends both for use in western blots...recombinant versions of mouse monoclonal antibodies generated and characterized by the Geiss Lab in their effort...vectors for microglia was further enhanced by packaging the virus as self-complementary AAV (scAAV). However...to single-strand AAV (ssAAV). To maintain the packaging capacity and improve the specificity of ssAAV,...

  4. Viral Vectors 101: Viruses as Biological Tools

    Type
    Blog Post
    Published
    March 21, 2023, 1:15 p.m.
    ...accommodate a cargo up to approximately 8 kb, but viral packaging is less efficient when inserts are on the higher...non-integrating) as lenti and retro viruses do. They are generally used to achieve transient, high expression of ...Gene Expression Infects Non-Dividing Cells Large Packaging Capacity (>8kb) High Transduction Efficiency ...Tropism for Mouse Cells and Hematopoietic Cells Low Packaging Capacity (~4.5kb From basic science to clinical... 4. Cronin J, et al. “Altering the tropism of lentiviral vectors through pseudotyping.” Curr. Gene. Ther... AAV Figure out How to Get the Most from Your Lentiviral Transduction Additional Resources on Addgene.org...viruses used in mammalian research: 𝛄-retrovirus, lentivirus, adenovirus, and adeno-associated virus (AAV)...

  5. 5 Tips for Troubleshooting Viral Transductions

    Type
    Blog Post
    Published
    Aug. 11, 2016, 2:23 p.m.
    ...contaminating your transduction with the packaging cells. Packaging cell debris can be removed either by ...you can check if virus is being generated by your transfected packaging cells. This check can be performed... particles in a smaller volume. Always remove packaging cell debris before proceeding to ultracentrifugation...step (5 minutes at 300-500 g). After removing packaging cell debris, virus can be pelleted by ultracentrifugation...reduce the volume of cell culture medium on the packaging cells immediately following transfection. In other...fold (Stockschläder et al., 1999). 5. Check your packaging cells for infection If your transductions aren...disrupting the flow of your experiment. If your packaging cells have been successfully transfected and produced...

  6. New Tools Enable CRISPRa for Neuroscience Applications

    Type
    Blog Post
    Published
    July 2, 2019, 12:51 p.m.
    ...can be delivered to the CNS by lentivirus, which has a larger packaging capacity than AAV. Such an approach...Schematic of the Cre-dependent SPH construct for generating the SPH mouse. (B) AAV plasmids used to express...should be screened for activity in vitro and then packaged into AAV for in vivo delivery. Multiplex gene ... also possible and is only constrained by AAV packaging limits (i.e., how many sgRNA expression cassettes...by the Day Lab, who developed neuron-optimized lentiviral vectors for in vitro and in vivo CRISPRa (Savell...gene expression. Gene isoforms refer to mRNAs generated from the same gene but with different untranslated...regulation — at least, more straightforward than generating cDNA for each gene isoform individually. To highlight...

  7. Getting Started with Behavioral Neuroscience: Standardizing Experiments and Using Viral Vectors

    Type
    Blog Post
    Published
    Aug. 20, 2019, 12:31 p.m.
    ... behavioral studies. General considerations for behavioral testing As a general consideration for behavioral... required. On the other hand, lentiviral vectors are superior packaging vectors for larger genes - they...mostly astrocytes. Lentiviral vectors are pseudotyped in most cases with VSV-G that generally mediates broad...brain region of interest. AAV or lentivirus?  Both AAV and lentiviral vectors are used for intracerebral...given some time to adapt to a testing apparatus. General stress levels of the test animal may also be reduced...vector should be used? Modifying gene expression Generally speaking, one may want to achieve either inhibition...injection. Find more details about choosing AAV or lentiviral vectors in our previous blog posts.  Viral vector...

  8. Viral Vectors 101: Pseudotyping

    Type
    Blog Post
    Published
    April 20, 2021, 2:30 p.m.
    ... (3) the packaging plasmid. These are transfected into target cells which produce lentiviral particles... rabies glycoprotein into the lentiviral envelope allows the lentiviral vector to be transported retrograde...viral vector, you use the same protocol for generating lentivirus, but you use a different envelope glycoprotein...related to lentivirus, many retroviral envelope proteins have been pseudotyped on lentiviral particles.... Quick Guide to All Things Lentivirus Get tips for titering your lentiviral preps Resources on Addgene.org...Addgene.org: Check out the lentivirus guide Find ready-to-use lentiviral preps at Addgene ...To deliver genes using lentiviral vectors, you need an envelope protein on the virus’s surface and a ...

  9. Four Ways to Package Transgenes That Exceed the Size Limit of Adeno-associated Virus

    Type
    Blog Post
    Published
    Sept. 1, 2020, 1:15 p.m.
    ... its packaging capacity is limited to ~4.7kb, or roughly half the packaging limits of lentiviral and adenoviral... up packaging an oversized genome of ~9 kb into AAV. They found that in defiance of the packaging limit...determined that even when larger vector genomes are packaged, the physical size of the genomes is still ~4.7kb...oversized AAV genomes are fragmented or truncated when packaged and then following transduction, the partial genomes.... Each piece of the gene is then independently packaged into an AAV. When a cell is co-infected with both...full-length transgene (Chamberlain et al., 2016). It’s generally accepted that some form of homologous recombination...exact mechanism is not yet known. Drawbacks:  Generally an overlapping split vector has equal or higher...

  10. Plasmids 101: The protein expression toolbox

    Type
    Blog Post
    Published
    Feb. 16, 2023, 7:05 p.m.
    ...integrate your choice cDNA. Both lentivirus and retrovirus can package cDNAs for genomic delivery and have...CRISPR-Cas9 and a custom gRNA is all you need to generate a frameshift mutation that will permanently ablate...transiently induce expression? Check out Addgene's Lentiviral dCas9 SAM vector and Piggybac dCas9 SAM vector...promoters and modes of action Check out Addgene's Lentiviral Tet-on and AAVS1 targeted Tet-on transgene vector...Adapted from Nabet et al. Check out Addgene's Lentiviral dTAG cloning vector and SMASh degron tagging ...Addgene website Promoter Guide CRISPR Guide Lentiviral Guide  ...

  11. CRISPR 101: Epigenetics and Editing the Epigenome

    Type
    Blog Post
    Published
    June 24, 2020, 5:45 p.m.
    ...cellular reprogramming of iPSCs, more efficient. In general, epigenetic chromatin modifications are correlated...modifications to DNA or histones, the proteins that help package DNA, were associated with gene expression or silencing...Core, as well as pLV-dCas9-p300-P2A-PuroR for lentiviral expression. Figure 1: dCas9-p300 adds ...decrease methylation and activate transcription. A lentiviral vector with the same modifier, Fuw-dCas9-Tet1CD...Tet-dependent (TetO-dCas9-D3A) constructs. For lentiviral expression, Fuw-dCas9-Dnmt3a and Fuw-dCas9-Dnmt3a-P2A-tagBFP...hUbC-dCas9-MQ1(Q147L)-EGFP is also available for lentiviral transduction. Lysine-specific Demethylase 1 (...particularly large protein, and this fusion allows for packaging into viral vectors and targeted delivery into ...

  12. Viral Vectors 101: AAV Serotypes and Tissue Tropism

    Type
    Blog Post
    Published
    Jan. 16, 2025, 3:42 p.m.
    ... 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...and can be selected as a serotype for Addgene’s Packaged on Request service. Some AAV1 tools are available...and can be selected as a serotype for Addgene’s Packaged on Request service. Some AAV2 tools are available...central nervous system, including neurons, AAV5 is generally the best choice for targeting astroglia. It has...and can be selected as a serotype for Addgene’s Packaged on Request service. Some AAV5 tools are available...and can be selected as a serotype for Addgene’s Packaged on Request service. Some AAV8 tools are available...

  13. Hot Plasmids: Spring 2025

    Type
    Blog Post
    Published
    June 3, 2025, 1:15 p.m.
    ...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...

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

    Type
    Blog Post
    Published
    July 12, 2021, 1:15 p.m.
    ...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...(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...

  15. Choosing Your Perfect Empty Backbone

    Type
    Blog Post
    Published
    Aug. 19, 2014, 3:39 p.m.
    ...remember that you will need accessory packaging vectors to generate the virus. Bacterial vectors This system...vivo (try pcDNA3-EGFP). If you can get away with generating transiently transfected cells, you will not need...basic mammalian expression system and go for a lentiviral or a retroviral backbone. This is considered ...

  16. Viral Vectors 101: Types of viruses

    Type
    Blog Post
    Published
    June 6, 2023, 1:15 p.m.
    ...-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 ...interchangeable between the two systems, other, more general, viral components such as a heterologous envelope...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...

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

    Type
    Blog Post
    Published
    Oct. 23, 2018, 11:49 a.m.
    ...analyses needed for packaging AAV. AAV production Overview: The first step in packaging AAV is transfecting...had never worked with AAV, so naturally I had to package some virus for my first experiment. It was a bit...transfecting HEK293 cells with AAV packaging plasmids. The cells need three different plasmids to produce AAV...intermediate into the single-stranded genome that's then packaged into AAV’s protein shell, or capsid; 2) the pHelper...plasmid which encodes a transgene of interest that’s packaged into the virus. Two to five days after transfection...plasmids contain ITRs which are required for proper packaging of AAV, but also form secondary structures that...spreadsheet similar to this one to plan my AAV packaging PEI transfections. It saves me time and helps ...

  18. How to Deposit Your Plasmids with Addgene

    Type
    Blog Post
    Published
    Feb. 27, 2024, 2:15 p.m.
    ...categories (e.g., for a lentiviral vector to be used in mammalian cells, check Lentiviral and Mammalian Expression...process, simply enter your preprint title or a general description of the deposit. Select your desired...millions. Pooled libraries must be verified by next-generation sequencing. Please see the help center article...that can be used under BSL1 conditions were generated in your lab are necessary for use of a plasmid...be prone to recombination in bacteria) such as lentiviral, retroviral, and AAV plasmids, we recommend the...with a unique Deposit ID. Addgene will send you a package containing instructions on how to prepare your ...

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

    Type
    Blog Post
    Published
    Sept. 12, 2017, 1:44 p.m.
    ...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 ...

  20. Addgene: The First Twenty Years

    Type
    Blog Post
    Published
    Dec. 19, 2024, 2:15 p.m.
    ...channel 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...
Showing: 21 - 40 of 54 results