... with Cas9-expressing lentiviruses will no longer be carried over to the gRNA lentiviruses. This means...stable Cas9-expressing cell line and then transduce this line with lentiviruses expressing various gRNAs...requested lentivirus and AAV of 2016. The top viruses of 2016 were (drumroll please)... Top Lentivirus: lentiCas9...single-vector systems typically express Cas9 and a gRNA from the same plasmid. LentiCas9-blast virus can be...lentiCas9-blast lentivirus from Addgene, you could use it in conjunction with gRNA lentiviruses easily made...single-vector system). Tips for using this plasmid Cas9 expression is typically cytotoxic, which makes generation...and transgene expression across different cell types (Aschauer et al., 2013). Expression of hM4D(Gi)-mCherry...

...four different plasmids into the producer cells (two packaging plasmids, an envelope plasmid, and the lentiviral...retroviruses and lentiviruses the same thing? Can I package lentivirus with retroviral packaging plasmids (and vice... of lentiviral plasmids created for a variety of applications including cDNA expression, shRNA-mediated...majority of transfer plasmids use the weak viral LTR promoter to drive expression of the gene of interest...levels of protein expression tends to be much lower than those seen when using a plasmid backbone that was...generation systems consist of an envelope plasmid and a packaging plasmid encoding HIV proteins gag, pol, rev...generation systems require an envelope plasmid, and two packaging plasmids, one encoding gag and pol and a second...

...function. Lentivirus/gamma-retrovirus Lentiviruses are a class of retrovirus (so all lentiviruses are retroviruses...lentiviral plasmids Addgene's most popular retroviral plasmids Addgene's collection of AAV plasmids Addgene...split across several plasmids (often three), for safety purposes. The primary plasmid with the delivered...often a gene) is called the transfer plasmid while the other plasmids with packaging or replication components... but not all retroviruses are lentiviruses.) This means that lentivirus and gamma-retrovirus share a lot...similar, and is contained on the transfer plasmid. If expression is the goal, a promoter should flank this...what will be delivered and expressed in the host cell from the transfer plasmid. Viral packaging and entry...

...of producing CRISPR lentivirus. As we’ve heard from scientists, producing lentivirus is not always an easy... order ready-to-use lentivirus from Addgene, you’ll also receive transfer plasmid DNA that you can use...along with lentiviral packaging plasmids to create your own lentivirus stocks. Our research scientists...rough approximations of the total number of CRISPR plasmids for that particular organism available at Addgene...helped distribute many of these technologies in plasmid form and are proud to have facilitated their fast...fast adoption. However, in many cases the plasmids themselves are only the starting point for the production... things in science, getting a high titer Cas9 lentivirus prep takes a bit of time and effort that, for...

...requested plasmid of 2017 is the lentivirus envelope plasmid pMD2.G from Didier Trono’s lab! Lentivirus envelope...example (the top requested plasmids from 2015 and 2016 were CRISPR plasmids), but so are lentiviral vectors....G This plasmid has been requested nearly 6,000 times (over 800 orders in 2017) and expresses the VSV-... producing our ready-to-use lentivirus preps. You can find our lentivirus production protocol on our website...titering your lentivirus in another blog post. If you need a quick primer on how lentiviruses work, check...top requested plasmid of 2018! Additional Resources Quick Guide to All Things Lentivirus 5 Tips for Troubleshooting...Plasmid technologies are constantly evolving, but sometimes a technology is so useful it forever enhances...

...packaging plasmids as well as transfer plasmids. This concept ensures safe handling and expression of these...cloning and expressing your gene of interest with GFP expression.  Cloning and expressing RNAi pMKO.1 ...separating viral genes and transgene expression across several plasmids. The most commonly used viral vector... lentiviral envelope plasmids are listed here. Please note that both lentiviruses and gamma-retroviruses...gamma-retroviral packaging plasmids are not interchangeable. General envelope plasmids, such as VSV-G, may be...gamma-Retroviral Transfer Vectors   Expressing your gene of interest pMKO.1 GFP (Plasmid #10676) Retroviral vector.... CRISPR technology pdCas9-humanized (Plasmid #44246) Expression of a catalytically inactive (dead), human...

...genome and stable expression of the transgene   Transfer plasmids The transfer plasmid is the vector you...virus (MoMLV) and consisted of two plasmids: the packaging plasmid containing all the structural genes...crossover event between the vector plasmid and the packaging plasmid caused the Psi sequence to be placed... facilitates nuclear export) is expressed from a separate plasmid, and the promoter of the 5’LTR has been...(tTA, also expressed by your transfer vector) binds to the TRE and activates the expression of your gene...usually 293T cells, with your transfer plasmid and your packaging plasmids. For this, you can use the transfection...Research Order Ready-to-Use Lentivirus Browse Our Lentivirus Guide Pages  ...

... multiple plasmids. Second generation packaging systems use 3 plasmids; an envelope plasmid that usually...encodes VSV-G, a packaging plasmid containing structural genes and a transfer plasmid carrying the transfer...Rev gene from the packaging plasmid and incorporating it into a fourth plasmid. In addition, the promoter...and packaging plasmids in order to obtain the best titers. Requiring an additional plasmid for transfection...Producing lentivirus: it's all about the T's (transfection and titers) To produce lentivirus, the plasmids...producing lentivirus, it is time to titer. One important consideration when titering a lentivirus is the ...Resources on Addgene.org Lentiviral Guide Find lentivirus plasmids Browse all viral vectors ...

...γ-retroviral packaging plasmids are not interchangeable. General envelope plasmids, such as VSV-G (Vesicular...in trans by specific plasmids. This method, therefore, reduces the number of plasmids that are required ...encoded by the transfer plasmid, but instead are provided in trans by other plasmids or the packaging cell...do many γ-retroviral plasmids have similar names? The nomenclature of the plasmid backbone is meant to...components are needed: γ-Retroviral transfer plasmid encoding a transgene, sgRNA, or shRNA of interest..., which facilitate integration of the transfer plasmid sequences into the host genome. Typically, it is... genome upon viral transduction. Most transfer plasmids available from Addgene were derived from MoMLV...

...Stable Cell Lines with Lentivirus Generating Stable Cell Lines with Lentivirus You may also like... Viral... the most desirable level of expression. Over time, transgene expression in a polyclonal population may...Protocol to generate stable cell lines expressing a gene of interest from an integrated lentiviral vector...protocol can be used to generate stable cell lines expressing a gene of interest from an integrated lentiviral...lentiviral vector. Unlike the short-term protein expression observed using transient transfection approaches... lentiviral vectors enables long-term protein expression studies. Moreover, repeating experiments in a...Corning 3516 15 mL conical tubes, VWR 21008-216 Lentivirus preparation Appropriate antibiotic for selection...

...consisted of three plasmids: the packaging, envelope, and transfer plasmids. The packaging plasmid carried a mutated...requested plasmids are used with lentiviral expression systems. If you’re new to the world of lentivirus, check... packaging and envelope plasmids are two of Addgene’s most requested plasmids! Lentiviral particles with... make stable transgene-expressing or knockout cell lines. Many CRISPR plasmids are designed for lentiviral...useful viral vectors in the lab. Advantages of lentivirus include a large genetic capacity and the ability...into the genome, permitting long-term transgene expression, but they could only infect actively dividing...non-dividing cells, but without sustained transgene expression. To design a viral vector system that could do...

... with immortalized cell line cultures. Lentivirus Lentivirus is a type of retrovirus and shares many ...-studied lentivirus is HIV, which many lentiviral tools have been derived from. Lentiviruses have slightly... commonly used lab viruses: gamma-retrovirus, lentivirus, adenovirus, and adeno-associated virus. Gamma-retrovirus...integrate into proto-oncogenes and disrupt their expression (Robinson, 1982). To add insult to injury, retroviruses...gamma-retrovirus, coming in around 9 kb. Unlike gamma, lentivirus can infect both dividing and non-dividing cells...nucleus as gamma-retroviruses do. Fun fact: Since lentivirus and gamma-retrovirus are so closely related, ...their required components are very similar. While plasmids containing virus-specific elements such as the...

...three plasmids: (1) The transfer plasmid, (2) the envelope plasmid, and (3) the packaging plasmid. These...Pseudotyping is only done on enveloped viruses such as lentivirus, retrovirus, and rabies virus. How to pseudotype...vector, you use the same protocol for generating lentivirus, but you use a different envelope glycoprotein...pseudotyped protein is encoded on the envelope plasmid which contains a promoter, the envelope gene, and... lentiviral particles.   Why pseudotype? Lentiviruses and retroviruses typically target immune cells...virus, HIV-2, and human foamy virus in cell lines expressing galactosyl(alpha1-3)galactosyl (alphaGal) sugars...than viruses produced in cell lines that do not express these sugars. (Takeuchi et al., 1997). The alphaGal...

...your virus and assaying for expression of a gene carried on the transfer plasmid or quantifying the number... transfer plasmid was used in the transfection; if preparing several different lentiviruses in parallel...Resources on the Addgene Blog Read Our Lentivirus FAQ Learn How Lentivirus is Used in CRISPR Genome-Wide Screening...prepped your DNA, transfected and harvested your lentivirus. Now it’s time to move ahead with your infection... that comes from amplification of the original plasmid which can over-inflate results. A packing vector...that are positive for vector encoded transgene expression following transduction with serial dilutions ...antibiotic resistance genes carried on transfer plasmids. Target cells are transduced with serial dilutions...

...for using lentivirus, infect and select, seems simple, in actuality, many find using lentivirus to be time...Lentiviruses are a powerful laboratory tool often employed to establish cell lines that stably express...quiescent cells do not take up nucleic acid or express transgenes as well as actively dividing cells resulting...compete with host cells for nutrients and can alter expression of receptors, ion channels, and growth factors...infection, happy virus is the second. When harvesting lentivirus, some labs prefer to collect and pool the harvests...after transfecting with the transfer and packaging plasmids. A benefit of the multiple harvest strategy is...on a variety of factors such as the transgene expressed, the viral storage buffers used, and other experimental...

...achieved with lentivirus but offer an improved safety profile, customization, and enhanced expression in many... while both AAV and lentivirus have low immunogenicity, AAV still beats lentivirus. In mice, AAV is almost...this case, the CRISPR components — are expressed. Unlike lentivirus, cargo delivered by AAV does not integrate... to use a multi-plasmid system, where you have one AAV vector dedicated to expressing Cas9, while the ...Addgene. The large packaging capacity and stable expression of lentiviral vectors make them an attractive...attractive option. However, safety requirements and expression limitations may be too constraining for some experiments...many mouse tissues. Safety AAV and lentivirus have different safety profiles that often affect which is ...

...or suggest additional plasmids with the Viral Service Suggestion Form. Lentivirus As some of the most popular... of our new Viral Service, we’re distributing lentivirus (with many CRISPR tools included among the preps...accelerate your research.   Why distribute virus? Plasmid distribution is booming, but, over the years, many... to knockout a gene, replace it, modulate its expression, or perform a genome-wide screen, we’ve got viruses... viruses to help you out. Of course, lentiviruses are used in experiments other than just those involving...involving CRISPR and so we also have lentivirus for delivering control shRNAs, GFP, and rtTA from the Tet-...experiments, while rtTA can be used to activate expression of a gene under the control of the tet operator...

...in a 1-plasmid system, in which Cas9 is included on the gRNA-containing plasmid, or a 2-plasmid system...generate lentivirus (B). Cas9-expressing cells or wild-type cells are treated with lentivirus containing...Quick Guide to All Things Lentivirus Get Tips on Titering Your Lentivirus Plasmids 101: NGS Quality Control...gRNA-containing plasmids. CRISPR genome wide-screening experiments commonly use lentivirus to deliver a ...contain thousands of gRNA plasmids, discerned only by a unique barcode on each plasmid. As such, sequencing...are composed of many different gRNA-containing plasmids, each with a different genomic target. The goal...a pooled population of gRNAs to Cas9 expressing cells. Pooled lentiviral CRISPR libraries (referred to...

...transfection of single plasmids vs pooled plasmids; for multiple edits, single plasmids are generally more...one vector to express the gRNAs and Cas9. Co-transfections can lead to variable expression levels due to...Multiplexed Lentiviral Expression Cassettes lentiviral transduction One vector expresses Cas9, eGFP, and ...Multiplexing crRNA expression with Cpf1  The key advantage of multiplexing crRNA expression with Cpf1 is that...vitro and when expressed in 293 cells. This allows for a single promoter to drive expression of a crRNA array...cleavage sites. This eliminates bulk from crRNA expression plasmids resulting in smaller, more streamlined vectors...transfected on a single plasmid. This is versus 2.4% of cells transfected with a pool of plasmids that contained...

...HEK-293 cells. When all of these viral plasmids are expressed within the production cell line, viral particles...integrated into the host genome? Expressed episomally long-term? Expressed short-term? Answering these questions...perform the transduction with: either retrovirus, lentivirus, adenovirus, or AAV. Then, your desired DNA to...viral particles are packaged, when the vector is expressed. Once the design and construction steps are complete...typically transfected along with viral packaging plasmids into a viral production cell line, usually HEK...Then the viral particles do the rest! Downstream expression or integration resulting from transduction typically...desired event.   Figure 1: Transduction to expression process. A viral particle enters a host cell,...

... 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 ...

... Protocols Lentivirus Production Lentivirus Production You may also ... 0.72 pmol Transfer Plasmid* 1.64 pmol OptiPro SFM to total volume 500 μL *Plasmid concentrations and ... each transfer plasmid. Pro-Tip Endotoxins can inhibit transfection; therefore, plasmid DNA purification...endotoxin removal step. For high quality plasmid DNA, the plasmids should also be propagated in an endonuclease... cm dish). Using transfer plasmid pHAGE TRE dCas9-KRAB (total μg of plasmid DNA 27.8 μg), this would be... 293T cells were transfected with the GFP-expression plasmid pRosetta using μg total DNA to μg PEI ratios...this protocol to generate lentivirus Protocols...

...could lead to reduced transgene expression over time, as the lower expressing clones take over the polyclonal... pool of stable cells. Transducing cells with lentivirus results in a heterogenous polyclonal population...that are more likely to retain stable transgene expression. Other methods of generating a monoclonal cell...protocol for generating stable cell lines with lentivirus . Day 0: (optional) Seed cells for the generation...protocol for generating stable cell lines with lentivirus . Pro-Tip Because the polyclonal cell pool is...population be monitored regularly for transgene expression. This protocol has been optimized for adherent...sufficiently expanded, screen the lines for transgene expression and/or other phenotypes. For example, perform...

...Pooled Libraries All Viral Plasmids Packaging Plasmids Transfer Plasmids Lentiviruses, a subset of retroviruses... Viral Plasmids Lentiviral Plasmids Lentiviral Plasmids Additional Resources...among multiple plasmids. Some plasmids (typically called packaging and envelope plasmids) encode components...packaging plasmids that can be used with 2nd and 3rd generation lentivirus technologies. ID Plasmid Generation...generation transfer plasmids Trono 12259 pMD2.G N/A Envelope VSV-G-expressing envelope plasmid Trono 8454 pCMV-VSV-G...envelope plasmid Nienhuis and Salmon 35616 pCAG-VSVG N/A Envelope VSV-G-expressing envelope plasmid Nienhuis...lentiviral plasmids to express your CRISPR gRNA? Please see our CRISPR Empty gRNA plasmids table and search...

...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...

...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...

...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)...

... Viral Vector Packaging Service Lentivirus Browse In-Stock Lentivirus Pooled CRISPR Libraries Cas9 Pooled...Barcoding Libraries Control Addgene's lentiviruses are made from plasmids that are currently in our repository... list of gRNA plasmids targeting specific genes. For more information on CRISPR plasmids and function,...library expresses EGFP for easy visualization via direct fluorescence. Morris Control Lentivirus Browse... of available viruses based on plasmids in our repository that express fluorescent proteins. ID Name Insert...Ready-to-use lentivirus available from Addgene's viral vector packaging service. Browse constructs currently...repository. You can obtain the plasmid from us or receive a ready-to-use viral preparation from the inventory...

...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...

...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%...