...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...

... 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...

...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...

...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 ...

...should be handled at BSL-2. AAV can transduce both dividing and non-dividing cells with a low immune response...expressing Rep2 and Cap1 James M. Wilson 104963 pAAV2/2 AAV2 AAV packaging plasmid, expressing Rep2 and Cap2...e.g., pHelper) and specific packaging cell lines (e.g., HEK293 cells) during AAV production. Read our AAV...

...gene modification. Maeder ML et al. Mol Cell. . 2008. Jul 25. 31(2):294-301. PubMed PMID 18657511 . Oligomerized...OPEN OPEN gGTCATCTGCcattcGAGGCGGACa REST corepressor 2 OZ553 and OZ554 OPEN OPEN gCTCACCATCtgttggaTGCGATGGAa...

...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...

...NGG-3′ (“N” can be any nucleotide base, followed by 2 guanines). This limits the use of ABEs as DNA sites... in the promoter region of gamma hemoglobin (HGB1/2) leads to continuous expression of fetal hemoglobin...primary human cells  Cell therapy approaches can use base editors to correct mutations in stem cells and return...primary human cell editing, the researchers tested ABE8 in CD34+ hematopoietic stem cells. ABE8 efficiently... In addition to stem cells the authors targeted 6 genes in primary human T cells using 8 selected ABE8s...to edit primary human T cells efficiently makes it a promising tool for T cell therapy development.  Reducing...be introduced into cells as a plasmid or as mRNA constructs. In primary human cells mRNA delivery of ABEs...

...Regulation Ben Deverman 2:00 PM Cre-DOG and Combinatorial Strategies Connie Cepko 2:45 PM Break 3:00 PM (...GCaMP6f-P2A-nls-dTomato Detailed protocol: AAV Production in HEK293 Cells Please share materials by uploading to your Dropbox...