... for the transgenic construct). More importantly, the expression of this construct is Cre-dependent, enabling...Figure 1: (A) Schematic of the Cre-dependent SPH construct for generating the SPH mouse. (B) AAV plasmids...by the Day Lab, who developed neuron-optimized lentiviral vectors for in vitro and in vivo CRISPRa (Savell...neuronal physiology. The Day Lab’s neuron-optimized lentiviral vectors can be found here. High-throughput screening...Cas9 activators can be delivered to the CNS by lentivirus, which has a larger packaging capacity than AAV...Savell et al., 2019). This dual lentivirus system pairs ubiquitous expression of sgRNA(s) and mCherry (... such as EF-1α and CAG. Figure 2: Dual lentivirus system with ubiquitous expression of sgRNA (top...

...Blog Learn More about Lentivirus Figure out How to Get the Most from Your Lentiviral Transduction Additional... individually overexpressed and available for lentiviral packaging and delivery to target cells as a pooled...with each cell receiving only 1 TF expression construct. Cells were cultured following a differentiation... Fig. 1: Barcoded TF library was packaged in lentiviral particles and used to transduce human embryonic...

...started, here are a few key details. Our set of constructs comprises all SARS-CoV-2 viral proteins except...viral protease, Nsp5. Our set also includes a GFP construct as a control. For easier portability, all viral...projects should be fun and straight-forward. All constructs are tagged with a 2xStrep-tag for easy detection...vector background, so you can package them into lentiviral vectors if you need to. Additionally, they have...correctly-sized proteins by Western Blot for 25 constructs, and confirmed expression via mass spec for two...Krogan Lab SARS-CoV-2 plasmids (Note: The Nsp6 construct is undergoing QC and will be available shortly...

...flexibility. Find the engineered prime editors here A lentiviral CRISPR knockout library that targets human epigenetic...the scale of your CRISPR screens by using this lentiviral CRISPR human genome-wide library deposited by...library delivers 2 randomly paired guides per construct and allows a reduced scale in screens without ...serotypes are now available for Gordon Fishell Lab constructs targeting GABA neurons. Serotypes include AAV1...

... well as the viral LTRs, differ. As a result, lentiviral and γ-retroviral packaging plasmids are not interchangeable...using 293T cells This method is very similar to lentiviral packaging methods. The three components described...wildtype LTR promoter is no longer present in these constructs. Why do many γ-retroviral plasmids have similar...found within both the 5' and 3' LTRs of retro/lentiviral plasmids. U5; Unique 5'; region at the 5' end... cells a virus can infect. Note: While both lentiviruses and γ-retroviruses use the same gene products...How do engineered γ-retroviruses differ from lentiviruses? There are a few important differences between...between engineered γ-retroviruses and lentiviruses. First, these viruses were derived from different genomes...

...generate negative control constructs — more on this later. For more on constructing fusion proteins, check...makes it easier to generate a large number of constructs for high-throughput screening of PPIs. For example...generally involves three steps: fusion protein construction, cellular expression, and quantification (Kerppola...described in more detail below: Fusion protein construction Fusing FP fragments to your PIPs requires careful... Connection. Cellular expression Whether BiFC constructs are expressed transiently by transfection or ... comparing the expression levels between BiFC constructs and their endogenous counterparts using western...blotting. The subcellular localization of BiFC constructs should also be compared against those of their...

..., 2018). From this data, they generated lentiviral constructs using the High-Throughput Mutagenesis and...carrying the corresponding wild-type genes were constructed as well. They called this library the CTD2 FASMIC...

...Howarth lab created the MBPx-SpyCatcher construct. This construct is used to produce a maltose binding protein...However, the laborious construction of repair templates and guide RNA constructs, as well as the difficult... Human Kinase Domain Constructs Kit, a library of human kinase domain constructs developed through a large-scale...the authors packaged it into lentivirus and used the resulting lentiviral library to generate a population...GMAP-compatible backbones for lentivirus LV 1-5 and retrovirus RV 2-5 were constructed. The authors then used...Science. 2013. PubMed PMID: 23328393 Wu lab human lentiviral CRISPR library     CRISPR pooled libraries enable... that results in the creation of many E. coli constructs with greatly varied levels of expression. The...

...available from Addgene. These plasmids enable lentiviral expression of N- or C-terminally FKBP12F36V-tagged...detailed information on cloning procedures used to construct the deposited plasmids. Molecules: We synthesized...unclear which terminus should be tagged, the dTAG lentiviral plasmids can serve as a rapid means of comparing...multiple FKBP12F36V-tagged proteins using our lentiviral expression platform, to assess selective degradation...

...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...produced 90% accurate integration of multiplexed constructs into target genomes and a 10-fold improvement...

.... Next, consider the expression level of your construct: would you like to stay as close as possible to...Finally, you can add bells and whistles to your construct. If you are planning to track your protein in ...basic mammalian expression system and go for a lentiviral or a retroviral backbone. This is considered ...

...small but growing catalog of ready-made AAV and Lentiviral preps. This new Viral Service represents Addgene...and New England Biolabs sponsored the capital construction of our new BL2+ laboratory and the infrastructure...establish consistent, high-quality protocols for lentiviral production. Bryan Roth’s lab from the University...the Addgene Blog Check Out the Top Requested Lentivirus and AAV of 2016 Learn How You Can Use the Viral...

... CRISPR pooled libraries have been created in lentiviral systems for mammalian screens, which enables ...for screening of cells that are refractory to lentiviral transduction, now enabling forward genetic screens... voltage changes in worms and have made these constructs available at Addgene. These include Arch(D95N...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...

...plasmid and the baculovirus construct, called a bacmid. Like AAV and lentiviral packaging plasmids, bacmids...-293T cells. If you titrate your viral preps, lentiviral titration requires an additional couple of days...require cloning specific AAV or lentiviral machinery into a baculovirus plasmid. Production of baculovirus...cells Viruses AAV and lentivirus Baculovirus; can produce AAV and lentivirus if needed # of plasmids...Overcoming the Challenges of Lentivirus Production Tips for Titering Your Lentivirus Preps More resources on...fast-growing, and widely available. The viruses Lentiviruses, AAV, and adenoviruses can all be produced in...cells. However, it is much more common to make lentiviruses and AAV in-house. High-titer adenoviruses are...

...reverse transcriptase, protease, and Bst polymerase constructs from their assay to Addgene.  Drew Endy’s and...19 pandemic, the Krogan lab at UCSF generated lentiviral vectors expressing SARS-CoV-2 open reading frames...all but one are cloned into pLVX vectors (for lentiviral packaging) and contain an IRES-Puro marker for...(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...

...Brunello CRISPRko sgRNA library as pooled library or lentiviral prep! Dolcetto: human CRISPRi sgRNA library While...the Dolcetto library! Now also available as a lentiviral prep. Calabrese: human CRISPRa sgRNA library ...allowing the screen to be conducted with only two constructs (the other containing dCas9-VP16). Like CRISPRi...function. Find the Calabrese pooled library and lentiviral prep here! Applications for the Root and Doench...

...MSCV-IRES-Luciferase IRES Retroviral pWPI IRES Lentiviral AmCyan-P2A-mCherry P2A Mammalian pC5Kan-P2A...-P2A P2A Insect pUltra P2A and T2A Lentiviral Ac5-STABLE2-neo T2A Insect   For the co-expression...same multicistronic vector, effectively making a construct that expresses multiple unique genes at equivalent....) PubMed. 3. Highly Efficient Multicistronic Lentiviral Vectorswith Peptide 2A Sequences. Ibrahimi et...

...infecting cells with sgRNAiBAR library containing lentiviral vectors the iBARs can be detected by next generation...multiplicity of infection (MOI) during cell library construction to ensure that the majority of cells receive...truncating one of the recombination sites in the construct to different lengths, the density of cells that...

...generating a series of single- and double-tagged constructs (including "CLAP", combining an N-terminal CLIPf... expression and labeling approach using these constructs in HEK 293 cells (Smith et al., 2024). They then... license.  The team deposited an array of constructs for tagging the kinesins KIF5A and KIF5B as well...Kv2.1 targeting motif as well as a pan-membrane construct without the Kv motif. Altogether, if you want ...proportions from a vector backbone that allows lentiviral packaging as a single payload (Figure 4). ...

...One good system to balance these needs is the lentiviral-based expression of floxed dCas9 so that it may... for cells stably and highly expressing these constructs can be achieved by fluorescence-activated cell... stable gRNA expression can be achieved using lentiviral backbones expressing blasticidin or puromycin...