...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...when you turn off expression for good, the Cre-Lox system offers inducible yet permanent gene excision. Essentially...introduction of the Cre recombinase. This is a time-tested system but does require knock-in of the LoxP sites, which...transcriptional activators fused to a dead Cas9. The system is led, via a custom gRNA, to your gene of interest...transiently induce expression? Check out Addgene's Lentiviral dCas9 SAM vector and Piggybac dCas9 SAM vector...On/off control all the time If your experimental system requires tunable control - or maybe you’re just...

...Instead, viruses replicate by hijacking the host’s system to produce more virus and continue the infection...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...

... (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... EnvA-TVA receptor-envelope system (Wickersham et al., 2007). This system requires first supplying rabies...pseudotyping of lentiviral vectors enables retrograde axonal transport and access to the nervous system after peripheral...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...

...Casilio-ME, which is based on their Casilio system. This system allows for targeted delivery of Tet1 alone...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...uncharacterized enhancers. The popular TALEN-based LITE system, which uses light to regulate transcription, also...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... targeted site compared to other Tet1 delivery systems. Transcriptional repression DNA Methyltransferase...

... 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...handling animals Experiment Behavioral scoring systems (experimenter differences in behavior observation...vector should be used? Modifying gene expression Generally speaking, one may want to achieve either inhibition...

... 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... (2008) A Hybrid Vector System Expands Adeno-associated Viral Vector Packaging Capacity in a Transgene-independent...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...

...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...

...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...

...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...

...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...

... 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...

... 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...

...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...

...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...

...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...

... 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...

...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...

...? 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...

...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 ...

... 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...