...determine what ratio gives the highest percentage of GFP positive cells. Refer to the table below for a possible...Data Figure 1: 293T cells were transfected with the GFP-expression plasmid pRosetta using μg total DNA to...efficiencies as measured by the highest proportion of GFP positive cells without limiting cell growth. Left...Left panels: bright field images; right panels: GFP channel images. Last reviewed on: August 2, 2023... Protocols Lentivirus Production Lentivirus Production You may also ...This protocol can be used to produce lentivirus from a lentiviral vector transfected into 293T cells using...this protocol to generate lentivirus Protocols...

...cells optimized for viral production (AAVpro or Lenti-X), but any HEK293T cell line should work. This ...determine what ratio gives the highest percentage of GFP positive cells. Refer to the table below for a possible...checking for protein expression. Sample Data Legend: Lenti-X 293T cells were transfected using 1:1, 1:2, 1:...

...containing the gene of interest (pTet-IRES-EGFP, pPRIME-Tet-GFP-FF3) and one with either tTA or rtTA (FUW-M2rtTA...viral titre to the number of cells transduced by a GFP encoded virus, and the reverse transcriptase activity...property makes the lentivirus an attractive choice for a gene transfer vector. Lentiviral vectors Although...generation of lentiviral vectors consisted of three plasmids encoding 1) the lentiviral vector genome ...used. Inducible lentiviral transfer vectors The most widely used inducible lentiviral vector system is...Your Research Lentiviral Vector Uses and Overview Resources on Addgene.org Find Lentiviral Vectors for...Ready-to-Use Lentivirus Browse Our Lentivirus Guide Pages  ...

...Purpose PI 17446 pLenti CMV GFP Hygro (656-4) GFP Hygromycin 3rd gen lentiviral eGFP expression vector...dCAS9-VP64_GFP dCAS9 (D10A, H840A) none Expresses dCAS9-VP64 activator with 2A GFP Zhang 61425 lenti dCAS-VP64...2A-Blast-WPRE) Zhang 61426 lenti MS2-P65-HSF1_Hygro MS2-P65-HSF1 Hygromycin lenti vector encoding the MS2... dCAS9 (D10A, N863A) Blasticidin 3rd generation lenti vector encoding dCAS9-VP64 with 2A Blast resistance... Viral Vector Packaging Service Lentivirus Browse In-Stock Lentivirus Pooled ...sgRNA library Brunello in lentiGuide-Puro Human sgRNA library in backbone lentiGuide-Puro targeting 19,114...gRNA pooled library in lentiCRISPRv2 Human sgRNA library in backbone lentiCRISPRv2 targeting 19,114 genes...

...hUbC-driven EGFP; can be used for cDNA expression Baltimore 12247 pLVTHM 2nd EF-1a-driven GFP and shRNA ... a reporter. See plasmid 17618 for GFP plasmid. Cheng 17452 pLenti CMV Puro DEST 3rd Gateway destination...gRNA plasmids table and search "lenti". ID Plasmid Generation Description PI 8453 pLKO.1 puro 3rd U6-driven...Blog: Tips for Titering Your Lentiviral Preps Lentiviral Pooled Libraries All Viral Plasmids Packaging Plasmids...researcher safety. Read our Lentiviral Guide for more information about: Lentiviral components 2nd versus 3rd...generation lentiviral systems Viral production Frequently asked questions about lentivirus use Glossary...Glossary of commonly used lentiviral terms Popular Lentiviral Envelope and Packaging Plasmids One safety feature...

...from BRAF-edited and GFP-edited amplification products (BRAF-GFP duplex) and from GFP-edited amplification... for gene editing. A virus encoding GFP plus a gRNA targeting GFP (pXPR_011, 59702-LV) was used to assay... by the virally-delivered GFP gRNA, will edit out the virally-delivered GFP gene. So, cells with no Cas9...I thought I could see GFP expression with a fluorescence microscope, but the GFP expression was so low...expressed GFP) that I couldn’t get a good image. You could probably also do Western blotting against GFP expression...amplification products (GFP-GFP duplex) as a control. 4. After optimizing the reaction and confirming the presence... didn’t. 2. In the original publication they see GFP expression in about 20% of their Cas9-expressing ...

...released Anti-GFP [N86/8R] as another option for targeting GFP. But why might you need a second GFP antibody...PMCID: PMC9015817.   Meet anti-GFP [N86/8R]! by: Ashley Waldron Anti-GFP [N86/38.1R] was among the first... targeting system Heat-inducible lentiviral Car constructs Anti-GFP [N86/8R] ready-to-use antibody Hyperfolder...western blot for mEGFP-HRas (from Plasmid 18662). See the Applications section on the Anti-GFP [N86/8R] page...?  Similarities:  Generated against full length GFP and converted to recombinant form by the NeuroMab...variable domains that recognize different epitopes on GFP Monoclonal antibodies can yield relatively low signal... details. Image credit: Addgene.   Find anti-GFP [N86/8R] here!   Andrews NP,  et. al. A toolbox of...

...respectively of a GFP dependent Cre. The two segments come together in the presence of GFP to form a functional... activation to cells expressing both Cre and GFP. The GFP dependent FLPo, Flp-DOG (75469), is also available...targeted expression DREADDs GFP dependent Cre and FLPo And more!  New lentiviral preps SARS-CoV-2 protein...in new serotypes, as well as a new Cre dependent GFP control plasmid for expression in forebrain GABA-...Karl Deisseroth 83895 AAVrg pAAV-hDlx-Flex-GFP-Fishell_6 Gordon Fishell   Optogenetics Optogenetics...transgenic reporter lines that supply a variety of GFP expression patterns, thus allowing you to further...AAV2 pAAV-Ef1a-fDIO mCherry 83900 AAVrg pAAV-mDlx-GFP-Fishell-1 Biosensors Plasmid Serotype Name ...

...The 2A-GFP allows for screening of transfected cells. Find pSpCas9(BB)-2A-GFP (PX458). lentiCRISPR v2 -...lines already expressing Cas9. Find lentiGuide-Puro. pSpCas9n(BB)-2A-GFP (PX461) - Similar to pSpCas9(BB)...requested plasmid of 2017. Find pMD2.G. pSpCas9(BB)-2A-GFP (PX458) - This plasmid from the Zhang lab expresses...BB)-2A-Puro (PX458), pSpCas9n(BB)-2A-GFP (PX461) expresses both a Cas9 protein and a gRNA. But instead ...nick, rather than cleave DNA. Find pSpCas9n(BB)-2A-GFP (PX461). pSpCas9(BB)-2A-Puro (PX459) - This mammalian...promoter. It’s a 3rd generation lentiviral backbone that must be used with lentiCas9-Blast or with cell lines...the last 15 years - the majority of these being lentiviral or CRISPR tools. How many times have these plasmids...

...Vectors Lentivirus Oka Lab Lentiviral Vectors Baylor College of Medicine Gene Vector Core Lentiviral and ...promoter pCDH-EF1-copGFP 73030 Expresses copGFP from the EF1 promoter pCDH-EF1s-Nluc-P2A-copGFP-T2A-Puro 73032...pCDH-EF1s-copGFP 73034 Expressed copGFP from a truncated EF1 promoter pCDH-CMV-Nluc-P2A-copGFp-T2A-Puro...Resources Addgene Blog: Your Lentiviral Plasmid FAQs Biosafety Resource Guide Lentiviral Transfer Vectors Adenoviral...deposited a series of lentiviral and adenoviral transfer vectors with Addgene. Lentiviral Transfer Vectors ...infectious virus. Protocol: Lentivirus Transfer Vector Production Protocol Lentiviral Vector ID Purpose pCDH-EF1...pCDH-EF1-DIO-copGFP 72253 Expresses copGFP under EF-1 promoter when Cre is expressed by another vector pCDH-EF1...

... out a pair of genes pXPR_219 Violet-excited GFP (vex) Rosa26-Cas9 I-CHIME Inducible knockout...knockout of a single gene pXPR_068 Violet-excited GFP (vex) Rosa26-FlpO-ERT2; H11-Cas9 L-CHIME ...types (lineage-based) pXPR_070 Violet-excited GFP (vex) Lineage-Cre; Rosa26-Cas9 S-CHIME Knocks...of genes sequentially pXPR_220 Violet-excited GFP (vex) Rosa26-FlpO-ERT2; H11-Cas9 Table 1: X-CHIME...(Rosa26-Cas9) hematopoietic stem cells with a lentiviral vector containing a gRNA. These transduced stem...systems which utilizes custom mouse strains and lentiviral expression vectors (Table 1). The four systems...pXPR_071, a plasmid that leads to higher titer lentivirus preparations than the first generation CHIME ...

...dim GFPs The availability of the three dimensional structure of GFP, its brighter mutant S65T-GFP, and...and GFP shRNA. Mice carrying the R26DsRedR; CRUSH-GFP; and Nestin-Cre alleles showed efficient GFP knockdown...identified a family of GFP proteins in the cephalochordate Branchiostoma floridae, named bfloGFPs. They went on...and demonstrate that they can successfully create GFP fusion proteins with a variety of genes across the...enabled scientists to engineer a wide variety of GFPs with diversity in fluorescence brightness, intensity...Cas9 gene editing. Dr. Masahito Ikawa has created a GFP reporter plasmid for scientists to validate the efficacy...plasmid undergoes HR or SSA to reconstitute functional GFP, and the cells will turn green. Mashiko et al.,...

...technology CAG-GFP-IRES-CRE (Plasmid #48201) This retroviral vector from the Gage lab encodes GFP and Cre-recombinase...Vectors   Expressing your gene of interest pMKO.1 GFP (Plasmid #10676) Retroviral vector for cloning and...and expressing your gene of interest with GFP expression.  Cloning and expressing RNAi pMKO.1 puro (Plasmid...descriptions of the lentiviral elements and the 2nd and 3rd Generation Lentiviral Systems can be found...found in our Lentiviral guide. Popular packaging and lentiviral envelope plasmids are listed here. Please...pre-treated with a compound of interest. Lentiviral vectors Popular Lentiviral Transfer Vectors    Expressing your...3rd generation lentiviral vector backbone from the Shih lab. You can find more lentiviral expression vectors...

...array plus a GFP tag. Four weeks post-infection, there was strong expression of Cpf1 and GFP in the targeted...4 targets when an array of 4 crRNAs, Cpf1, and a GFP tag were transfected on a single plasmid. This is...~75% of neurons were co-transduced with Cpf1 and GFP.  Of these neurons, ~15% had indels at all 3 targeted...Multiplexed Lentiviral Expression Cassettes lentiviral transduction One vector expresses Cas9, eGFP, and ...expressing the Cpf1 multiplex system from a lentiviral vector.  Lentiviruses carry a (+) strand RNA copy of the... options for Cpf1 multiplexing: Transfection, lentivirus, and AAV Cpf1’s ability to process its own pre-crRNA... multiple expression platforms: transfection, lentiviral transduction, and AAV transduction.  In most ...

... pCAG-SpCas9-GFP-U6-gRNA 79144 Mammalian hU6 yes, cut S. pyogenes EGFP Zou pCAG-eCas9-GFP-U6-gRNA 79145...S. pyogenes Puro Zhang lenti sgRNA(MS2)_zeo backbone 61427 Mammalian/Lentiviral BsmBI none S. pyogenes...Mammalian/Lentiviral LIC none S. pyogenes GFP Vakoc U6>sgRNA(F+E) 59986 Other/Ciona intestinalis BsaI none S....Yang pLCKO 73311 Mammalian/Lentiviral hU6 none S. pyogenes Puro Moffat lenti sgRNA(MS2)_puro backbone 73795...pyogenes URA3 Davis lenti sgRNA(MS2)_puro optimized backbone 73797 Mammalian/Lentiviral U6 none S. pyogenes...cut Lb Cpf1 Neo Welker AIO-GFP 74119 Mammalian U6x2 yes, nick S. pyogenes EGFP Jackson AIO-mCherry 74120...Mammalian/Lentiviral BbsI none S. pyogenes Puro, TagBFP Yusa lentiGuide-Puro 52963 Mammalian/Lentiviral BsmBI...

... later, Cre-dependent GFP expression was detected with direct fluorescence. GFP was not detected in the...viral genomes (vg)/cell, pAAV-CAG-FLEX-rc [Jaws-KGC-GFP-ER2] (Addgene 84445-AAVrg) alone at 1.1E6 vg/mL, ...prep # 55632-AAVrg). pAAV-CAG- FLEX-rc [Jaws-KGC-GFP-ER2] was a gift from Edward Boyden (Addgene viral...Browse our AAV vector collection. Lentivirus Production Lentiviral preparations ("-LV” catalog items)...Concentrated lentiviral preparations (“-LVC” catalog items) are generated from lentiviral preparations...being frozen. Specifically, lentiviral particles are collected from the lentiviral preparation by precipitation...for in our reported titers. Lentiviral vectors are titered using a lentiviral droplet digital PCR (ddPCR...

.... https://doi.org/10.1101/788679 A destabilized GFP reporter for calcineurin activity in yeast Article...reporter consisting of a quickly-degraded destabilized GFP variant fused to a 4X CDRE repeat. In addition to... Ca2+ (bottom panels) equipped with destabilized GFP calcineurin reporter (blue) and stained with PI (...death. Image from Diessl et al., 2020. Find the GFP reporter calcineurin activity here Diessl et al.,...infecting cells with sgRNAiBAR library containing lentiviral vectors the iBARs can be detected by next generation... They created plasmids for purification of tTALE-eGFP and tTALE-mCherry. In comparison to intercalating...

...both the GFP and the RFP proteins in the nucleus. During the first round of translation, the GFP proteins...Compartment mCherry-Rab5 BFP-Rab5 GFP-Rab5B Vacuolar & Budding Compartment GFP-Rab4B Mitochondria mito-BFP...GTPase in division) GFP-Mff (outer membrane protein) Late Endosome mCherry-Rab7A GFP-Rab7A     ...protein Knock-off, is made up of three components: a GFP protein that binds to the coding region of a reporter...reporter mRNA via a PP7 coat protein (NLS-PCP-GFP) a RFP protein that binds to the 3’ UTR of the reporter...these dCas9s with one of three fluorescent proteins (GFP, BFP and mCherry), the authors were able to visualize...monoclonal antibodies against them, fused them to GFP, and compared them to popularly used epitopes tags...

...includes a set of plasmids each containing an anti-GFP nanobody fused with a different functional element...using different detection methods. Since the anti-GFP nanobody is able to bind fluorescent proteins derived...here can be used to target any protein fused with GFP, YFP, mCerulean, or other related fluorophores. Find...specific DNA sequence in only 1 min. Using these lentiviral plasmids, catalytically inactive Cas9 (dSpyCas9...

... create a fusion with a reporter protein such as GFP? All of these outcomes can be most effectively achieved...typically used for gRNA. May contain reporter gene (e.g. GFP) to identify and enrich positive cells or a selection...transfer vectors. May contain reporter gene (e.g. GFP) to identify and enrich positive cells. Likely contains...efficiency. Lentiviral transduction Cas9 and gRNA can be present in a single lentiviral transfer vector...plasmids provide the necessary components to make lentiviral particles. Stable, tunable expression of Cas9...Difficulty Very easy Very hard Viral delivery   Lentivirus Retrovirus AAV Cells Dividing or nondividing...: Traffic Light Reporter System: Can evaluate lentiviral component delivery as well as genome repair by...