...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...

...46, JNK1, JNK1A2, JNK21B1/2, PRKM8, SAPK1, SAPK1c MAPK9 JNK-55, JNK2, JNK2A, JNK2ALPHA... PRKM11, SAPK2, SAPK2B, p38-2, p38Beta MAPK12 ERK3, ERK6, P38GAMMA, PRKM12, SAPK...Mitogen-activated protein kinases (MAPKs), also known as extracellular signal-regulated kinases (ERKs), are serine/...threonine-specific protein kinases. They regulate diverse cellular programs by acting as an integration point for...

... mutations in the gene methyl-CpG binding protein 2 ( MECP2 ). MECP2 Rett syndrome is an X-linked disorder...X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat Genet . 23, 185–188. (Link opens in a new window... Cuddapah et al. 2014. Methyl-CpG-binding protein 2 (MECP2) mutation type is associated with disease severity... Specific mutations in methyl-CpG-binding protein 2 confer different severity in Rett syndrome. Neurology...males with mutations in Methyl-CpG binding protein 2. Am J Med Genet B Neuropsychiatr Genet . 180, 55–67...Unpublished Joost Gribnau Human Cell Line Models Induced pluripotent stem cells (iPSCs) are a powerful system...opens in a new window) LUHMES cell line , an immortalized pre-neuronal cell line that can be differentiated...

...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...

...different inherited genes: Presenilin 1, Presenilin 2, and APP gene.The majority (>90%) of individuals develop...study aggregation. Saha et al. Nat Commun. 2023 Feb 2. Study the C terminal domain of TDP-43 to better understand... rodent species. Boender et al. Sci Adv. 2023 Jun 2. Use Cas9 in astrocytes. Endo et al Science. 2022 ...with the iPSC toolbox . Lam et al. bioRxiv. 2022 Dec 2. Use PiggyBac plasmids with tet-inducible expression...protein-protein interactions and nerve cell function within the cell. Parkinson's Disease Parkinson’s disease...inducible vector . Harley et al. Cell Rep. 2023 Dec 26. Induce CNS cells with concomitant expression of ...with the disease and implicate a wide range of cellular processes. The vast majority of cases (90-95%)...

...Layer 2-3_IT Isocortex 230803 pAAV-AiE2638m-minBG-iCre(R297T)-BGHpA AiP20142 AiE2638m Cre Layer 2-3_IT ...Layer 2-3_IT Isocortex 220727 pAAV-AiE2543m-minBG-iCre(R297T)-BGHpA AiP2048 AiE2543m Cre Layer 2-3_IT ...Layer 2-3_IT Isocortex 230402 pAAV-AiE0680m-minBG-iCre(R297T)-BGHpA AiP15072 AiE0680m Cre Layer 2-3_IT ...to target a variety of cell populations in different brain regions. Labeled cell populations were tested...to primate neocortex. Cell Rep. 2021 Mar 30;34(13):108754. doi: 10.1016/j.celrep.2021.108754. PMID: 33789096...regulatory sequences — termed enhancers — to drive cell type-specific expression. The Allen Institute for...differential chromatin accessibility across different cell types and cloned into an AAV backbone. Most enhancers...

... Morris CellTag Barcode Library Version 2 Ready-to-use lentiviral particles carrying version 2 of the ...the CellTag barcoding library. Version 2 of the Celltag library contains 4934 barcodes to combinatorially...carrying version 1 of the CellTag barcoding library. Version 1 of the Celltag library contains 19973 barcodes...barcodes to combinatorially index cells for single-cell analysis of clonal dynamics. This library expresses...combinatorially index cells for single-cell analysis of clonal dynamics. This library expresses EGFP for easy visualization...carrying version 3 of the CellTag barcoding library. Version 3 of the Celltag library contains 5737 barcodes...

...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...

... 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 ...

...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...

... Podcast Interview with Stem Cell Researcher Darrell Kotton 2. Stem cell scientists care about patients...explained that it took 50 people nearly 2 years to create the first stem cell line that could be used in clinical...prevent stem cell lines from becoming the new HeLa cells?), genome engineering and stem cells, and the difficulty...opportunity to see Shinya Yamanaka celebrate the tenth anniversary of his landmark Cell paper showing that it was...generate induced pluripotent stem cells (iPSCs) capable of forming nearly all cell types in the body from otherwise... happening. Stem cells scientists have certainly not lost site of that. 3. Stem cell science is hard, ...otherwise terminally differentiated cells simply by expressing four proteins Oct4, Sox2, Klf4, and cMyc...

...Seed individual cells in a 96-well plate Day 2–14: Monitor cells for growth and expand cells Day 14–30: Analyze...colony size. Figure 2: Cas9 expression in monoclonal cell lines generated from A549 cells transduced with ... CO 2 incubator Pipet controller Hazardous waste container Microscope Hemocytometer or other cell counter... is about 2 × 10 6 cells in a 10 cm dish. Each 10 cm dish should be seeded in 10 mL DMEM complete, which...transgene expression ( Figure 2 ). Sample Data Figure 1: Generation of monoclonal cell lines from expansion of...Biological Safety Cabinet 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel...of a 5 cell/mL solution. Calculate the total cells needed: Total cells needed: 10 mL × 5 cells/mL = 50...

... 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...

...surviving cells. Even in the absence of cell death, the cell media should be changed every 2–3 days to...Day 0: Seed and transduce Cells Day 2–3 (am): Remove media, replace with fresh media containing selection...into two 75 cm 2 flasks, etc. Pro-Tip This selection method results in a polyclonal cell population, meaning...Safety Cabinet 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel... 200–1000 µL single channel pipette Ice bucket CO 2 incubator Pipet controller Hazardous waste container... the cell media. Cell death by some cells in the culture may adversely affect the surviving cells in the...media to make the cell solution in this step. To seed the cells: Prepare a batch of cells as follows: Dilute...

...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...

...recombining shuttle plasmids Vogelstein 16401 pAdEasy-2 Adenoviral For recombining shuttle plasmids; for use...transgene under CMV promoter Vogelstein 16407 pAdEasy 2-GFP beta-gal Shuttle Test plasmid that contains β-...target a broad range of dividing and non-dividing cell types with almost 100% efficiency. Unlike lentiviruses...recombination of adenovirus plasmids in either mammalian cells or microorganisms, including bacteria and yeast....can then be transfected into mammalian packaging cell lines to generate adenovirus particles. Read our...

...phage display. J Immunol Methods. 2000 Aug 28;242(1-2):159-81. Carlos Barbas Vector system for expression...+J phagemid libraries. Immunol Lett. 2015 Oct;167(2):95-102. Joanna Bereta Sybody Generation Toolbox for...expressing these in bacteria, mammalian cells, yeast, plants, or insect cells. Benefits include consistency between... research purposes include animals or hybridoma cells, but are difficult to generate due to expense and...monoclonal antibodies (R-mAbs) expressed in mammalian cells. Monoclonal antibodies are derived from an individual...transiently transfected into mammalian tissue culture cells, the R-mAbs are secreted into the culture media ...

...the specimen.  2. Fluorescence Activated Cell Sorter (FACs) A machine that sorts cells according to whether...photostability of your fluorescent protein when using 2-photon microscopy as this hasn't been well reported...PMID: 18703742. PubMed Central PMCID: PMC2606057. 2. Chtanova, Tatyana, et al. "Dynamics of neutrophil...labeling T cells, B cells, and antigen presenting cells, scientists have been able to decypher cellular dynamics...tracked by cell sorter giving immunologists the ability to count cells and statistically analyze cell movements...fast T cells could jump from one antigen presenting cell to another. Indeed, we now know that T cells are...2016)? How do dendritic cells migrate to lymph nodes to activate T cells (Celli et al 2008, Kitano et al...