... to Top A. pLKO.1-TRC Cloning Vector A.1 The RNAi Consortium The pLKO.1 cloning vector is the backbone...use of this vector. A.2 Map of pLKO.1 pLKO.1 is a replication-incompetent lentiviral vector chosen by the...pLKO.1 TRC-cloning vector. Addgene Plasmid Description pLKO.1 – TRC control Negative control vector containing...into the pLKO.1 vector, producing a final plasmid that expresses the shRNA of interest. C.1 Recommended ...pLKO.1 TRC Cloning Vector The following steps were the prevailing guidelines when the pLKO.1 vector was...digests. Digest pLKO.1 TRC-cloning vector with AgeI. Mix: 6 μg pLKO.1 TRC-cloning vector (maxiprep or miniprep...of Contents A. pLKO.1-TRC Cloning Vector A.1 The RNAi Consortium A.2 Map of pLKO.1 A.3 Related plasmids...

...lentiviral expression of OSKM reprogramming factors: Mouse Expression Vectors Human Expression Vectors... plasmid-based expression requires serial transfection of 1 or 2 plasmids that express the reprogramming... residual expression piggyBac Excisible Medium Medium Generates transgene-free and vector-free cells...harmful viral particles that express oncogenes, such as Myc. Viral vectors also have the largest genomic...so either a single polycistronic vector or multiple single-gene vectors can be used to package reprogramming...reprogramming. Additionally, lingering expression or re-expression of viral transgenes in iPSC-derived cells... retroviral vectors. This makes it possible to use a polycistronic cassette that expresses multiple reprogramming...

...transgene expression. Once packaged in AAV, this pre-recombined DNA could lead to transgene expression in recombinase-negative... their AAV vectors to find the optimal dose that allows for sufficient transgene expression but limits...blog Check out our Viral Vectors Featured Topic Page Subscribe to Viral Vectors Blog Posts Learn How We...animals, producing misleading results.  To date, we have analyzed hundreds of viral vectors and estimate that... of poor reagent quality. To make sure our AAV vectors are of the highest quality, we undertake a rigorous...To make sure that adeno-associated viral (AAV) vectors available at Addgene are suitable for in vivo studies...serotype. Additionally, we check viral transgene expression in transduced cells whenever possible. This post...

...Volume of 1 mg/mL PEI (μL) 1:1 18.9 18.9 1:2 18.9 37.8 1:3 18.9 56.7 1:4 18.9 75.6 1:5 18.9 94.5 1:6 18.9...protein expression. Sample Data Legend: Lenti-X 293T cells were transfected using 1:1, 1:2, 1:3 and 1:6 µg.... Thawed aliquots should be discarded after 1–2 months. 1 mg/mL polyethylenimine, linear MW 25,000 Da ...stable. Dilute 1:3 (µg DNA:µg PEI) in 500 µL total of OptiPro SFM (per 10 cm plate). 56.7 µL of 1 mg/mL PEI...µg of pRosetta :µg of PEI. The 1:2 and 1:3 ratios provided high transfection efficiencies as can be seen...General Transfection You may also like... Viral Vector Guides Virus Blog Posts Mol Bio Protocols Viral...polyethylenimine. Transfections allow for transient expression of a gene of interest in a target cell line and...

...easier to clone, express, and purify compared to antibodies. Nanobodies are easily expressed in the cytosol...#140702) The pMAK461-64 plasmids were expressed in SHuffle T7 Express competent E.coli (NEB) using IPTG induction...approaches rely on continual expression of both dCas9 and the gRNA. This expression can vary significantly ...fluorescently labeled piggyBac vectors for uniform and sustained expression of single or multiplexed gRNAs...and track cells expressing both dCas9 and gRNAs. The multi-gRNA piggyBac vector integrates into ... the mCitrine gene. mCherry is constitutively expressed. Cells are then cultured and FACS sorted. Image...Histone 2A or 2B can result in bright targeted expression while minimizing non-specific labeling. For labeling...

...Fluorescent Protein Plasmids Find Mammalian Expression Vectors ... spread out onto the plate. We test the (capped) 1:1 agarose/DMEM solution against a part of the body ...that overexpress genes of interest; more recently we have also been using Addgene CRISPR vectors to generate... enzyme assays to select both over- and under-expressing clones. If GFP-encoding cDNA is co-transfected...and PCR, depending on the level of endogenous expression. We highly recommend the use of an electronic...ampules each of 4 clones with different levels of expression of our gene of interest. Advantages of the agarose...isolate individual cell clones with different expression characteristics from a population of transfected...

...genome editing, activating gene expression, repressing gene expression, and much more. But there’s one... T, Lieber A (2018) HDAd5/35++ Adenovirus Vector Expressing Anti-CRISPR Peptides Decreases CRISPR/Cas9... means they can be delivered in vivo using AAV vectors. Having a way to shut off CRISPR-Cas systems prevents...cleavage (Harrington et al., 2017). Figure 1: Anti-CRISPR proteins (Acr's) can block CRISPR activity... ways to anti-CRISPR proteins in your experiments 1. Reduce off-target effects Prolonged Cas activity ...genes in the difficult-to-engineer Sulfolobus islandicus rod-shaped virus 2 (Mayo-Muñoz et al., 2018)....deletion could replicate when challenged with the S. islandicus CRISPR-Cas system. 5. Phage therapy Phage therapy...

...in viral vectors, but easily fit in a TE. Transposons are also less likely than viral vectors to induce...Transposons: The jumping genes. Nature Education 1(1):204. https://www.nature.com/scitable/topicpage/transposons-the-jumping-genes...Non-autonomous transposons, however, need another TE to express a transposase or reverse transcriptase so they ...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...

...Douglas Kim , GENIE Project Calcium Bicistronic vector expressing GCaMP6 and mRuby2 for imaging neural activity.... Nat Commun. 2018 Oct 25;9(1):4440. Eric Schreiter Calcium AAV expression of photoconvertible fluorescent...accumulation in bacteria. Biotechnol Biofuels. 2008 Jun 3. 1(1):11. Wolf Frommer Maltose Green fluorescent MBP-based... Huebener , Tobias Rose Calcium Soma-targeted expression of jGCaMP8f, GCaMP7f, or GCaMP6f Precision Calcium...6. Edward Boyden Calcium Intersectional viral expression of GCaMP6m/s in cells depdent on Cre, Flp, or... Dual- and Triple-Feature Intersectional Single-Vector Delivery of Diverse Functional Payloads to Cells...Karl Deisseroth , INTRSECT 2.0 Project Calcium Expression of GCaMP6f in PV cortical interneurons under ...

...General Design Figure 1: Two proteins are fused to the FP(11) and FP(1-10) fragments. When Protein...and 11 to yield large (1-10) and small (11) fragments. In several cases an FP(1-10) fragment may be compatible...Description Article PI Blue EBFP2 EBFP2(1-10) and Capri(1-10) for use with GFP(11) Multiplexed labeling...Commun. 2017 Bo Huang mNeonGreen3 mNG3K(1-10) and mNG3A(1-10) for use with mNG2(11) Improved yellow-...sfCherry2 sfCherry2(1-10) and sfCherry2(11), also photo-activatable variant PAsfCherry2(1-10) Improved split... vitro, and can be used for measuring protein expression or localization in cells, or even single-particle...Mol Syst Biol. 2014 Galit Lahav EYFP Gateway vectors with EYFP split at 175 Arabidopsis homolog of the...

...pitfalls: they require construction of plasmid vectors and/or rely on cumbersome in vitro synthesis of...purchased and there is no need to construct plasmid vectors or synthesize/purify IVT sgRNA in the lab. Figure... (although we could successfully knock-in non-expressed genes with similar efficiency, Dewari et al. 2018...tagging success, although many of these genes were expressed at really low levels so actual knock-in success...the tag sequence flanked by homology arms (Figure 1). In the first step of optimization, we started off...form the ribonucleoprotein complex (csRNP, Figure 1). We use in-house purified Cas9 protein, which, in...standard immunocytochemistry (ICC) assays.  Figure 1: Three ingredients of epitope-tagging experiments....

...Figure 2: iGluSnFR3 displays enhanced brightness, expression, and sensitivity compared with earlier SF-iGluSnFR...GEVIs. Some constructs are also available as viral vectors (here and here).  Figure 3: JEDI-2P was identified.... By fusing ITAM to an RTK of interest and co-expressing a cytosolic ZtSH2 reporter, they observe fluorescence...improved sensors for these and other targets. Figure 1: MTRIA biosensors are generated by inserting a circularly-permuted...fluorescent biosensors. Nat Commun. 2022 May 25;13(1):2919. doi: 10.1038/s41467-022-30685-x. Feel the ...performance for in vivo use and deep-tissue imaging. Vladislav Verkhusha’s lab has generated a new genetically...

...Example of viral targeting of cells expressing Cre AND Flp, here expressed through a double transgenic animal...recombinase-expressing mouse lines 2–9 and combinations of transgenic recombinase-expressing animal lines...Find intersectional expression plasmids that combine multiplexed recombinases, including Cre, Flp, and...and VCre to enable viral expression of molecular payloads in target populations defined by multiple parameters...understanding of neural circuit functionality. Precisely expressing these tools in well-defined cellular sub-populations... a single gene or projection). Intersectional expression approaches combine multiplexed recombinases, ... including Cre, Flp, and VCre to enable viral expression of molecular payloads in target populations defined...

...IGHDY1 IGHD1-1 immunoglobulin heavy diversity 1-1 IGHD11 IGHD1-14 immunoglobulin heavy diversity 1-14 (non-...IGHV6-1 immunoglobulin heavy variable 6-1 IGHV61, VH IGHV7-4-1 immunoglobulin heavy variable 7-4-1 IGHV7... TAPBP-R, TAPBPR THBS1 thrombospondin 1 THBS, THBS-1, TSP, TSP-1, TSP1 TRPC4AP transient receptor potential...variable 2-8 IGLV28, V1-2 IGLV3-1 immunoglobulin lambda variable 3-1 IGLV31, V2-1 IGLV3-10 immunoglobulin lambda...NCC-1, NCC1, SCYA13, SCYL1 CCL14 chemokine (C-C motif) ligand 14 CC-1, CC-3, CKb1, FLJ16015, HCC-1, HCC... 3 G0S19-1, LD78ALPHA, MIP-1-alpha, MIP1A, SCYA3 CCL3L1 chemokine (C-C motif) ligand 3-like 1 464.2, D17S1718...GPD, GpFy, WBCQ1 DEFA1 defensin, alpha 1 DEF1, DEFA2, HNP-1, HP-1, MGC138393, MRS DEFA3 defensin, alpha...