...values can be 10-1000 fold higher than for functional assays depending on the vector backbone. When using...primers that target the common features of viral vector backbones; once validated these universal primers...plasmid which can over-inflate results. A packing vector deficient in reverse transcriptase will produce...the number of target cells that are positive for vector encoded transgene expression following transduction... prep. This method is particularly useful when vectors carrying fluorescent markers are used as it eliminates...titration of pseudotyped HIV-1-based lentiviral vectors."Nature protocols 4.4 (2009): 495-505. PubMed PMID...Geraerts, Martine, et al. "Comparison of lentiviral vector titration methods."BMC biotechnology 6.1 (2006)...

...restriction enzymes or vector purchases (Table 1). Restriction enzymes and vector purchases can raise the...for the simple insertion of a PCR product into a vector. The time it takes to make a plasmid could increase...and very cool 8,768,560 hours – or almost exactly 1000 calendar years!   *Dammit, man, I’m a scientist...

...posts related to viral vector protocols and tips Read all of our viral vectors blog posts Resources on...Since Addgene began producing AAV vector preparations, we have used quantitative PCR to evaluate the ...used to calculate the viral titer: GC/mL = {[(R*C)(1000/V)]*D} R = Reaction volume C = Copies/uL V = Volume...evaluation of recombinant adeno-associated viral vectors." Human Gene Therapy ja (2019). PubMed PMID: 31140327...single-stranded and self-complementary adeno-associated viral vector genome titers by droplet digital PCR." Human gene...titration by qPCR Browse our collection of viral vector related protocols Learn more about the Addgene ...

... (response elements). Promoters are about 100 to 1000 base pairs long and found upstream of their target...we will discuss these in a future post on viral vector parts. Promoter specificity Aside from choosing... Can be induced by IPTG. Commonly found in pET vectors. Very tightly regulated by the lac operators. Good...-inducer fucose Weaker. Commonly found in pBAD vectors. Good for rapid regulation and low basal expression...Browse All Plasmids 101 Posts Learn about Mammalian Vectors Read about Reporter Gene like Luciferase and GFP...

...problems with Cas9 delivery. Adeno-associated viral vectors (AAV) are commonly used for in vivo gene delivery...SpCas9) and a gRNA together (~4.2 kb) into an AAV vector is challenging due to its packaging capacity of...packaged Cas9 and multiple gRNAs into separate AAV vectors, increasing overall packaging capacity but necessitating...includes SpCas9, and shortern orthologs approximately 1000 amino acids in size. From the pool of shorter orthologs...genome engineering: An adeno-associated viral (AAV) vector toolbox. Biotechnology Journal 9:1402–1412 . https...Editing: Strategies and In Vivo Delivery by AAV Vectors. Cell 181:136–150 . https://doi.org/10.1016/j.cell...

...into distinct expression vectors using the enzyme BbsI. These expression vectors each contain Type IIS restriction...Cas9 or dCas9-containing destination vectors. Destination vector options include humanized wt Cas9, dCas9...create lentiviral vectors expressing wt humanized Cas9 and up to three gRNAs. Entry vectors containing the...wt hCas9 entry vectors are supplied with the kit, you can clone your own entry vectors containing other...containing vector using Golden Gate assembly, and up to 8 gRNAs may be expressed simultaneously. Vectors for ...assembly of multiple components into a destination vector. The first step in CRISPR/Cas9 Golden Gate multiplexing...to allow for ordered assembly into a destination vector that expresses Cas9. This is illustrated in the...

...of template should have homology arms between 500-1000 bps. HDR efficiency with circular plasmid templates...plasmids that liberate the targeting region from the vector. Alternatively, PCR-generated or commercially synthesized...

...and in vitro methods are similar except that the vectors carrying the transdifferentiation factors are directly... (iPSCs) by temperature-sensitive Sendai virus vectors. Proc Natl Acad Sci U S A, 2011. 108(34): p. 14234...., Human induced pluripotent stem cells free of vector and transgene sequences. Science, 2009. 324(5928...mouse induced pluripotent stem cells without viral vectors. Science, 2008. 322(5903): p. 949-53. PubMed PMID...fibroblasts to myoblasts. Cell, 1987. 51(6): p. 987-1000. PubMed PMID: 3690668. 40. Ferber, S., et al., Pancreatic...Differentiate the Stem Cell Field Learn how lentiviral vectors can be used for gene delivery Resources on Addgene.org...Addgene.org Visit our Stem Cell Pages Find Viral Vectors for Your Research Find Plasmids from the Yamanaka...

...design 5 guide RNAs (gRNAs) targeting each of the 1000 genes you’d like to investigate in your next CRISPR...The gRNA oligos are subsequently cloned into your vector of choice using the adapter sequences that you ...Neville E., Ophir Shalem, and Feng Zhang. "Improved vectors and genome-wide libraries for CRISPR screening....

...expression vectors with various promoters suitable for in vivo expression, and/or produce vectors to make...prototrophic strains. The vectors were based on the original EasyClone vectors (Jensen et al 2013) allow...donor vectors containing mAID and either Neo or Hygro resistance markers with CRISPR/Cas vectors targeting... interest and cloned into provided destination vectors using low-cost, one-pot golden gate cloning that... Jacks Lab constructed several GMAP-compatible vectors for a wide variety of biological applications. ... problem, Csaba Pál’s lab has created a set of vectors that allows one to use the MAGE method in unmodified...clinically relevant bacterial species. pORTMAGE vectors open new horizons to modify genomes in a broad ...

...building block Entry vectors and 5 lentiviral Destination vectors. These vectors can induce combinatorial...research purposes, retroviral vectors have been developed into ‘split-vector systems’ by separating viral...and expression of these vectors when used in experiments. Retroviral vectors make up a large toolbox ...Gamma-retroviral vs. lentiviral vector systems Gamma-retroviral vectors have a relatively high transduction... many more useful viral vectors available at Addgene: Follow our viral vector blog posts for updates on...tips for using retroviral vectors in your experiments. Gamma-retroviral vectors Popular gamma-Retroviral...Transfer Vectors   Expressing your gene of interest pMKO.1 GFP (Plasmid #10676) Retroviral vector for cloning...

... of Gateway cloning vectors Vector type  Vector features Purpose Donor vector attP sites for recombination...sequences into multiple vector systems. With the appropriate entry and destination vectors, one can use Gateway...ccdB gene is present in the donor vectors and the destination vectors prior to recombination, and it is...donor vector.   Method B: TOPO-cloning of the desired insert into an attL-entry-TOPO vector. TOPO...Gateway vector to produce the desired expression clone. This is possible thanks to the Gateway vectors’ design...sites creates attL and attR sites.   Gateway vectors contain modified versions of the att sites so that...flanking the insert and the attP sites of the donor vector. This reaction is catalyzed by the BP Clonase enzyme...

...both vectors. Examples of use: Halbert et al., 2002 used an overlapping vector with 440 or 1000bp overlaps... overviews of three split vector strategies plus one additional split vector strategy for increasing the...homology directed repairs (HDR). Split AAV vector approaches Split vector approaches increase the size of the... hybrid vector, as measured by β-galactosidase activity, was 81% of that from a single vector, but significantly...ideal split vector system. Overall the efficiency of transgene expression from split vectors has been inconsistent..., Duan D (2008) A Hybrid Vector System Expands Adeno-associated Viral Vector Packaging Capacity in a Transgene-independent...) has many features which make it a great viral vector, but its packaging capacity is limited to ~4.7kb...

...recall, are the outer shell of an AAV vector, which determine the vector’s tropism, including its ability to...payloads. Since most systemic vectors are primarily derived from AAV9, the vectors have to be administered ...on the Addgene blog Viral Vectors 101: AAV Variables That Matter Viral Vectors 101: An Introduction to ...be suitable for your experiment. Like all viral vectors, the suitability of a systemic capsid for an individual...switches all take up precious cargo space in AAV vectors. Dual expression systems, which use two or more...administration (Chen et al., 2023). High viral titer High vector genome (vg) doses are recommended for all systemic...and 5 x 1011 vg per animal (i.e., 10 and 50 µL of vector at 1 x 1013 vg/mL titer), while AAV-PHP.S is typically...

...in viral vectors, but easily fit in a TE. Transposons are also less likely than viral vectors to induce...transposon’s LTRs, similar to when packaging viral vectors.   Let’s take a closer look at three different ... in human cells at rates similar to retroviral vectors. The hyperactive version of the SB transposase,...efficiency in human cells similar to retroviral vectors. The hyperactive PB transposase (hyPB) is ~10-fold...transfer Transposons are an alternative to viral vector delivery of transgenes, like for iPSC reprogramming... piggyBac, which is a big advantage over viral vectors (~5 kB payload for AAV and ~8 kB payload for lentivirus... out of the laboratory. F1000Res 9:135. https://doi.org/10.12688/f1000research.21018.1 Munoz-Lopez M, ...

...control the CRISPR system activity.  Using an AAV vector may help with size limitations. In addition, non-viral...& Parkinsonism. https://doi.org/10.4172/2161-0460.1000439 6. Wu et al. 2020. . “Advances in CRISPR/Cas-based...

...pGWF1 Cyan & Yellow Bacterial Gateway-compatible vector to express a gene of interest fused between ECFP...Bioessays. 2012 May; 34(5):341-50. doi: 10.1002/bies.201100098. Sekar RB, Periasamy A. Fluorescence resonance...

...more permanent knockdown, consider using shRNA vectors instead. With both siRNA and shRNA, it can be difficult...and flow cytometry. F1000Research, 13, 817. https://doi.org/10.12688/f1000research.154034.1 Laflamme, ...synaptotagmin 1 page. The data produced is available on the F1000 gateway and Zenodo community.  Knockdown approach...

...sequenced and quality controlled plasmids and viral vectors. When working with a plasmid from Addgene, you .... Finally there are new publishing outlets like F1000, PeerJ, PLoS, and bioRxiv that make it easier to...

... JA, Carnes AE, Hodgson CP. Plasmid DNA vaccine vector design: impact on efficacy, safety and upstream...Electrophoresis. 2010 Jul;31(14):2436-41. doi: 10.1002/elps.201000061. PMID: 20589859. Levene SD. Analysis of DNA topoisomers...