...splitting transfected cells into 3 plates at different densities for selection). A 2% agarose solution is...transition from a 48-well cell plate to a 96-well assay plate. We generally freeze at least 2 ampules each of 4...of agarose, which is then overlaid with the firmer 2% agarose gel solution discussed above for picking.... a wide variety of cell lines, from HEK to CHO cells to neural and endocrine cells such as AtT-20, PC12...then handled. Additionally, many cell lines, especially the endocrine cell lines we most commonly work with...specific cell line, and its transfection efficiency. We often trypsinize the transfected cells on the day...increased). For some cells, add a drop of serum to aid in outgrowth. Depending on the cell type, we sometimes...

...DSB and 2. a specific edit close to the gRNA PAM site. When a repair template is present, the cell may repair...Cas enzymes? CasPEDIA is an encyclopedia of Class 2 CRISPR systems with wiki entries describing enzyme...sequence. DSBs are preferentially repaired in the cell by non-homologous end joining (NHEJ), a mechanism...

...diphtheria toxin receptor fused to GFP for studying cell ablation. 2 Jessell , Azim 45580 pAAV-flex-taCasp3-TEVp...constitutive Expression of the tet-off transactivator (tTA) 2 Gradinaru 99120 pAAV-ihSyn1-tTA Inducible Synapsin...Scanziani Tools for Cell Ablation These AAV encode tools that can be used for cell ablation. ID Name Expression...Transactivators Affinity Purification Neurophysiology Cell Ablation Tetracycline Transactivators and Inducible...expression of designer pro-taCasp3 and TEVp for studying cell ablation. 5 Shah , Wells Don’t See What You’re Looking...

...AAV); 8–10 kb (lenti) 38 kb Cells HEK-293T Sf9 Time to collection 2–4 days 5–10 days Purification... R.M. (2002). Insect cells as a factory to produce adeno-associated virus type 2 vectors. Human Gene Therapy...Centrifugation Titration time ~1 day (AAV); ~1 week (lenti) 2–10 days Initial titers Higher Lower Resources...that already do mammalian cell culture. HEK-293T cells are the most common cells used in viral production...versus insect cells for viral production Consideration Mammalian cells Insect cells Viruses AAV ...type of cells that will be used to make the virus. Viruses can be produced in many different cell types,...produced in bacteria cells. However, some viruses can be produced in multiple different cell types, including...

...deletions in human cells using zinc finger nucleases. Genome Res. 2010 Jan;20(1):81-89. PubMed. 2. Bauer DE, ...with TALENs, in collaboration with the Porteus lab [2]. With the advent of CRISPR/Cas9, we began to explore...CRISPR plasmids into MEL cells by electroporation, sort your transfected cells via FACS, primer validation...article "Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9" from the Journal of Visualized...CRISPR/Cas9 to create genomic deletions in mammalian cell lines. Below Bauer and Canver discuss the motivations...deletions is to produce monoallelic deletion clones. Cells with just a single intact copy at a given locus ...screening by conventional PCR. Biallelically disrupted cells can be easily identified. Even in the case of clones...

... a new vial of early passage cells. Approximately 2 weeks post-thaw, cell culture supernatant is tested...item (see maps section for images). Figure 2: AAV Pro cells were transduced with either pAAV-Ef1a-mCherry-IRES-Cre...suggests that titers obtained using ddPCR are generally 2–3 fold higher than those achieved using the standard... preparations are added to cells in culture. Three to five days later, cell cultures are inspected for...involves transducing cells with serial dilutions of the lentiviral vector, harvesting cells several days later... (55634-AAVrg) alone at 1.7E6 viral genomes (vg)/cell, pAAV-CAG-FLEX-rc [Jaws-KGC-GFP-ER2] (Addgene 84445...Production Lentiviral preparations are produced in 293T cells using the 2nd generation psPAX2 and pMD2.G packaging...

...par with ZF arrays, if not slightly lower. Figure 2: Simplified representation of the Voytas/Bogdanove...variety of backbones in just a few steps ( Figure 2 ). The Bogdanove group also hosts web-based software...mammalian transcription. Nat Biotechnol. 2011 Feb;29(2):149-53. PMID: 21248753 . TALEs for the masses. Rusk.... Targeted gene disruption in somatic zebrafish cells using engineered TALENs. Sander JD, Cade L, Khayter...

...RNA-guided regulation of transcription in eukaryotes. Cell . 154(2):442-51. PMID: 23849981 Ishino Y, Shinagawa ...Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell . 163(3):759-71. PMID: 26422227 Zetsche...transcribed to make the pre-CRISPR RNA (pre-crRNA). (2) The pre-crRNA is processed into individual crRNAs...2015. Class 1 (Multi-subunit effector complex) Class 2 (Single multi-domain effector) Type I (Cas3) Type ... with unprecedented speed and specificity. Figure 2: An overview of CRISPR and NHEJ/HDR. The Cas9/gRNA...genomic loci in living human cells by an optimized CRISPR/Cas system. Cell . 155(7):1479-91. PMID: 24360272... for Mapping Multiprotein Complexes in Human Cells. Cell Rep . 13(3):621-33. PMID: 26456817 Fu Y, Sander...

...degrades the mRNA. RISC includes argonaute protein 2 (Ago2), which facilitates cleavage of the target. ... International Journal of Biomedical Science, 13 (2), 48–57. PubMed (Link opens in a new window) . Moore... in therapeutic settings. As a tool in mammalian cell systems, silencing is achieved through the delivery...). Origins and mechanisms of miRNAs and siRNAs . Cell, 136 (4), 642–655. PubMed (Link opens in a new window...

... genetically encoded H2O2 probe is pQE-30-HyPer3 (2) which he will co-express together with a plasmid ...Pharmacology 2017 Jun 15;134:1-17. PubMed PMID: 27984002. 2. Bilan, Dmitry S. et al. “HyPer-3: A Genetically Encoded... Accelerating science and supporting scientists globally are the shared mission of both Seeding Labs ...