...reprogramming factors Efficient germ-line transmission obtained with transgene-free induced pluripotent stem cells...Reprogramming Plasmids Differentiation and Transdifferentiation Plasmids Stem Cell Research Plasmids Stem...methods with the goal of helping readers choose a strategy suited to their research. Once you know which ...differentiated somatic cell into another via transdifferentiation. This method of cell lineage conversion ...technology. Browse Addgene's collection of Transdifferentiation Plasmids below. Many of the techniques for... for reprogramming, differentiation, and transdifferentiation utilize plasmids to increase expression ...human Klf4, Oct4, c-Myc, and Sox2 as VP-16 transcriptional activating fusions in single lentiviral vectors...

...introduce gRNA-targeted single-strand breaks in DNA instead of the double-strand breaks created by wild type... opposite strands of your DNA in close proximity. These double nicks create a double-strand break (DSB...dead dCas9 fused to a transcriptional activator peptide can increase transcription of a specific gene. ... BspQI Transfection Puromycin yes, cut Ji-Long Liu 45946 pU6-BbsI-chiRNA dU6 BbsI Transfection none, need...Genome Editing Cut Base Edit Nick Prime Edit Transcriptional Regulation Activate Interfere Epigenetics RNA...functional CRISPR/Cas enzymes will introduce a double-strand break (DSB) at a specific location based on a gRNA-defined...non-homologous end joining (NHEJ). Double nicking strategies reduce unwanted off-target effects. Nickase mutants...

...Plant transcription factors for Agrobacterium-mediated transient expression or plant transformation - Silin...parts for assembling plant transformation vectors - Daniel Voytas. Transcription Factors The following collections...expression in plants: Maize Transcription Factor ORFeome - John Gray. Rice Transcription Factor ORFeome - John...Plant Base Editors Save Time with Transient Plant Leaf Transformations RNA Interference in Plant Biology...Figshare - Plant Illustrations (Link opens in a new window) - Collection of illustrations and pictures of... Addgene Blog: Plant Biology CRISPR Cloning Transcription Factors RNAi Empty Backbones Fluorescent Proteins...Engineered biocontainable RNA virus vectors for non-transgenic genome editing across crop species and genotypes...

...sites are on separate DNA molecules (in trans), a translocation event is generated between the target sites...85577 pTC-CMV-Tet CreER expression and tetracyclin-dependent transgene/shRNA expression ApoE.HCR.hAAT Mammalian... pTC-ApoE-Tet CreER expression and tetracyclin-dependent transgene/shRNA expression ApoE.HCR.hAAT Mammalian...plasmids can be useful for lineage tracing, evaluating infection/transfection efficiency, selecting desired...of rearrangements: inversion, deletion, and translocation. Figure 1. Recombination outcomes are determined... If the target sites (loxP) are on the same DNA strand in opposite directions, recombination results in...reversed. B) If the target sites are on the same DNA strand (in cis) in the same direction, the sequence between...

...For easy-to-transfect cell types (e.g. HEK293 cells), transfection with standard transfection reagents may...mammalian cell line that can be transfected at high efficiency Lentiviral transduction Cas9 and gRNA can be present...present in a single lentiviral transfer vector or separate transfer vectors May contain reporter gene ...screens using CRISPR AAV transduction CRISPR elements are inserted into an AAV transfer vector and used to ...ribonucleoprotein (RNP) complexes are advantageous due to the transient Cas9 expression. The table below summarizes the...details, see Plasmids 101: Mammalian Vectors ) Transient or stable expression of Cas9 and/or gRNA in a ... mammalian cell lines Useful for difficult to transfect cell types, can also be used in vivo A common ...

... recoded strain of E. coli ( 68306 ) with the optimized phosphoserine orthogonal translation system ( ...Plasmid 69118 E17TAG GFP zeo resistance Strain 34929 BL21ΔserB Strain 52055 EcAR7 Plasmid 112031 pNAS1b split...phosphorylation is one of the most abundant forms of posttranslational modifications in cells and research into its..., Science 2013) and improving the orthogonal translation system to make singly or multiply phosphorylated...pSerOTS. Recently the Rinehart lab published a new strain of recoded E.coli (rEcoli XpS 192872 ) and an optimized... optimized phosphoserine orthogonal translation system (pSerOTS-C1* (V70) 188537 ). These reagents are...phosphorylation sites (“phosphosites”) consist of a central phosphoserine site flanked by 15 amino acids on...

...introduce gRNA-targeted single-strand breaks in DNA instead of the double-strand breaks created by wild type... opposite strands of your DNA in close proximity. These double nicks create a double-strand break (DSB...dead dCas9 fused to a transcriptional activator peptide can increase transcription of a specific gene. ...functional CRISPR/Cas enzymes will introduce a double-strand break (DSB) at a specific location based on a gRNA-defined...Genome Editing Cut Base Edit Nick Prime Edit Transcriptional Regulation Activate Interfere Epigenetics RNA...base editors convert C->T (or G->A on the opposite strand) within a small editing window specified by the...guanosine to create A->G (or T->C on the opposite strand) mutations. ID Plasmid Promoter PI Publication ...

...Tools Tetracycline Transactivators Affinity Purification Neurophysiology Cell Ablation Tetracycline Transactivators...These AAV encode tetracycline-inducible tools/controls and tetracycline transactivators that can be used...service encoding tet-off transactivators and tools for affinity purification (TRAP). Viral...used with tetracycline (tet)-inducible expression systems . ID Name Expression System Activity Serotype...CAG-driven, constitutive Expression of the tet-off transactivator (tTA) 2 Gradinaru 99120 pAAV-ihSyn1-tTA Inducible... promoter (ihSyn) Expression of the tet-off transactivator (tTA) with a positive feedback loop for amplified...ihSyn) Cre-dependent expression of the tet-off transactivator (tTA) with a positive feedback loop for amplified...

...- Tatjana Sauka-Spengler Lab Lineage Tracing Cell lineage tracing in multicellular systems typically relies...Lewis Lab PhOTO Zebrafish: A Transgenic Resource for In Vivo Lineage Tracing during Development and Regeneration...Periklis Pantazis Lab Multibow: digital spectral barcodes for cell tracing - Sean Megason Lab Zebrabow - Albert... (~3 months), ease of care and breeding, and transparency of the embryo and larvae, it is easy to culture...standard toolbox of genetic methods, such as Tol2 transposons, Cre-mediated site-specific recombination, morpholinos...emerged as a model system for human disease and translational research, including the study of cancer, inflammation...-Cas9 system - Shuo Lin Lab Re-purposed Ac/Ds transgenic system enabling CRISPR/dCas9-interference (CRISPRi...

...introduce gRNA-targeted single-strand breaks in DNA instead of the double-strand breaks created by wild type... opposite strands of your DNA in close proximity. These double nicks create a double-strand break (DSB...dead dCas9 fused to a transcriptional activator peptide can increase transcription of a specific gene. ...functional CRISPR/Cas enzymes will introduce a double-strand break (DSB) at a specific location based on a gRNA-defined...Genome Editing Cut Base Edit Nick Prime Edit Transcriptional Regulation Activate Interfere Epigenetics RNA...Base editors convert C->T (or G->A on the opposite strand) within a small editing window specified by the...guanosine to create A->G (or T->C on the opposite strand) mutations. CRISPR-X, a base-editing system from...

...diluting or concentrating to a final concentration of 0.9–1.1 mg/mL. Antibody concentration is confirmed...Blotting (WB) Proteins are extracted from cells, separated by SDS-PAGE, transferred to a membrane, and labeled...of co-purified plasmid from transfection. This residual plasmid is extracted and amplified with primers...expression vectors, human embryonic kidney cells are transfected with the plasmid(s) encoding the heavy and light... the buffer can be removed by dialysis or gel filtration. Titer and Purity Antibodies are initially titered...serial dilutions of a standard antibody of known concentration and stained with Coomassie blue. Protein band...standard curve is generated. The final antibody concentration is calculated from the standard curve. Using...

...introduce gRNA-targeted single-strand breaks in DNA instead of the double-strand breaks created by wild type... opposite strands of your DNA in close proximity. These double nicks create a double-strand break (DSB...dead dCas9 fused to a transcriptional activator peptide can increase transcription of a specific gene. ...Genome Editing Cut Base Edit Nick Prime Edit Transcriptional Regulation Activate Interfere Epigenetics RNA...functional CRISPR/Cas enzymes will introduce a double-strand break (DSB) at a specific location based on a gRNA-defined...base editors convert C->T (or G->A on the opposite strand) within a small editing window specified by the...guanosine to create A->G (or T->C on the opposite strand) mutations. ID Plasmid Gene/Insert Promoter Selectable...

...introduce gRNA-targeted single-strand breaks in DNA instead of the double-strand breaks created by wild type... opposite strands of your DNA in close proximity. These double nicks create a double-strand break (DSB...dead dCas9 fused to a transcriptional activator peptide can increase transcription of a specific gene. ...Genome Editing Cut Base Edit Nick Prime Edit Transcriptional Regulation Activate Interfere Epigenetics RNA...functional CRISPR/Cas enzymes will introduce a double-strand break (DSB) at a specific location based on a gRNA-defined...base editors convert C->T (or G->A on the opposite strand) within a small editing window specified by the...guanosine to create A->G (or T->C on the opposite strand) mutations. ID Plasmid Gene/Insert Promoter Selectable...

...Link opens in a new window) . Transfections are performed using the transfer plasmid, a plasmid encoding...are purified by iodixanol gradient ultracentrifugation and concentrated to purity and titers adequate for...cell cultures are inspected for sterility. Transducibility Some viral vectors are tested in vitro and ...section for images). Figure 2: AAV Pro cells were transduced with either pAAV-Ef1a-mCherry-IRES-Cre (55634...cleared by low-speed centrifugation and then by filtration through a 0.45 µm polyethersulfone (PES) membrane... been stored at -80 °C and thawed. Lentivirus titration is performed using a 293T cell line in the presence...using a lentiviral droplet digital PCR (ddPCR) titration protocol (modified from Wang et al. (2018) (Link...

...reverse transcriptase and integrase. Once in a target cell, the virus uses the reverse transcriptase in tow...long-term transgene expression. Some disadvantages of γ-retroviruses are that they can only transduce dividing...Kasparov Popular γ-Retroviral Transfer Plasmids Common γ-retroviral transfer plasmids. Want a retroviral...Plasmid for transgene expression; also expresses luciferase Lowe 9044 pMIG MSCV Plasmid for transgene expression...Addgene, including popular packaging, envelope, and transfer plasmids. Viral...of interest? Search our database for retroviral transfer plasmids that have inserts. ID Plasmid 5'LTR Description...resistance. Hahn 63704 pRetroX GFP T2A Cre CMV/MSV Tetracycline inducible expression of GFP T2A Cre fusion in...

...information on substrates and synthases needed for use of these tags. Plasmids Addgene ID Plasmid Substrate Feature...simultaneously with dual labeling when used with two substrates Can be used in live or fixed cells, and for internal... proteins and use enzymatic binding to attach substrate Control plasmids with a recognizable sub-cellular... is based on the human O6-alkylguanine-DNA-alkyltransferase (hAGT), a DNA repair protein, and is suitable...protein tags based on the acyl carrier protein. Substrates are derivatives of Coenzyme A (CoA). These tags...Vector SNAP-tag Empty backbone for stable and transient mammalian expression 101133 pSNAP-CaaX Control...Vector CLIP-tag Empty backbone for stable and transient mammalian expression 101134 pCLIPf-H2B Control...

...Adenoviruses have high transduction efficiency and direct high but transient transgene expression. γ-Retrovirus...virus for gene transfer in clinical trials. These viruses have a relatively high transduction efficiency,...Dividing/Non-dividing Yes Low AAV Transient or Stable* single stranded linear DNA ∼4.5 Kb 18-26 Dividing.../Non-dividing No* Very Low Adenovirus Transient double stranded linear DNA >8 Kb 105 Dividing/Non-dividing...are versatile tools because of their ability to transduce non-dividing cells. These viruses enable long-...immunogencity. These viruses can direct long-term transgene expression, but generally are not permanently ...permanently into the host genome, enabling long term transgene expression. Overview of Viral Plasmids While plasmids...

... of the CRISPR RNA (crRNA) and the trans-activating crRNA (tracrRNA; Hsu et al ., Nature Biotechnology...plasmids #61422 - #61424 were designed for transient transfection : 61422 : Expresses dCAS9-VP64 activator... cassettes to express the CRISPR RNA array and tracrRNA separately (Cong et al ., Science 2013). Additionally...or SpCas9n, D10A nickase) + CRISPR RNA array + tracrRNA: This plasmid contains three expression cassettes...human codon-optimized SpCas9; sgRNA can be co-transfected as PCR amplicons (Ran et al., Nature Protocols...references are below. Return to top Cas9 SAM transcriptional activation plasmids and screening library CRISPR...) is an engineered protein complex for the transcriptional activation of endogenous genes. It consists...

...easily and inexpensively generate R-mAbs by transiently transfecting the plasmids into mammalian tissue culture... Anti-Pan-Neurofascin (extracellular) [A12/18R] Pan-Neurofascin (extracellular) Rat Mouse IgG2a 177439...Anti-Kv1.4 K+ channel (extracellular) [L71/5R] Kv1.4 K+ channel (extracellular) Rat Mouse IgG2a 177489...Mouse IgG2a 177564 Anti-ADAM22 (extracellular) [N57/2R] ADAM22 (extracellular) Mouse Mouse IgG2a 177565 Anti-GluN2B...channel Rat Mouse IgG2a 188161 Anti-TRAAK K+ channel [N445/27R] TRAAK K+ channel Mouse Mouse IgG2a 188162...Anti-Kv1.4 K+ channel (extracellular) [L71/22] Kv1.4 K+ channel (extracellular) Rat Mouse IgG2a 190292...N382/14] MFF Human Mouse IgG2a 190320 Anti-TRAK2 [N390/43] TRAK2 Human Mouse IgG2a 190321 Anti-Navbeta2 Na...

...samples to extract proteins. Homogenize (tissues only), lyse, and then collect protein extract by centrifugation...hybridoma cells into plasmids. Plasmids are then transfected into cells and induced to express antibodies....to lot, as immune responses have high inter- and intra-individual variability. Other Affinity Reagents ...and therefore should be kept at 4 °C. Record and track lot numbers for all antibodies used in the lab. ...reaction in the presence of several different substrates. The luminescence produced by the reaction can... an SDS-PAGE gel to separate proteins by size. Transfer proteins from the gel to a membrane. Incubate ...and block the membrane. Activate the HRP with a substrate kit. Image the membrane using either X-ray film...