...heterogeneous population of mutant cells, with each cell or set of cells containing a mutation in a different...separately.  Mutant cells are enriched using either drug selection or fluorescence-based cell sorting and screened...mutant cells can be used in drug screens to identify genes that confer drug-resistance. Mutant cells are ...generate lentivirus (B). Cas9-expressing cells or wild-type cells are treated with lentivirus containing...plus Cas9 to generate the mutant cells, respectively (C). Mutant cells are screened (D) and "hits" are ...lead to skewed results. Selecting a cell type - Theoretically, any cell type can be used in a CRISPR screen...requires a massive amount of cells as starting material. Therefore, cell types that are of low abundance...

...). These cell-permeable Janelia Fluor (JF) dyes have excellent properties inside living cells, especially... fluorescent proteins. In-cell labeling strategies The majority of in-cell labeling strategies have two...and oligonucleotide labels for fixed-cell imaging, not for live-cell applications. Thus, these fluorophores..."Imaging live-cell dynamics and structure at the single-molecule level." Molecular cell 58.4 (2015): 644...sparked a renaissance in biological imaging. Suddenly, cell biologists were no longer beholden to chemists and.... Add a dash of DNA with an electrical jolt and cells become perfectly capable of synthesizing fluorophore...question: In this age of GFP and its ilk, why should cell biologists talk to chemists? One reason is something...

...enzyme-mediated proximity cell labeling strategy to detect cell-cell interactions in vivo (Ge et al., 2019...about RapID! EXCELL: Proximity labeling for the detection of cell-cell interactions EXCELL is an enzyme-mediated...et al. "Enzyme-Mediated Intercellular Proximity Labeling for Detecting Cell–Cell Interactions." Journal...a bait protein fused to BirA* in cells and then incubate those cells with exogenous biotin. After a few...-protein interactions, and even more recently cell-cell interactions. If you’re looking to identify interactions...proximal and interacting proteins in mammalian cells." J Cell Biol 196.6 (2012): 801-810. PubMed PMID: 22412018...peptide (LPETG). In EXCELL, the mgSrtA variant can be displayed on the surface of a cell of interest, where...

...restoring mCherry expression. Cells can then be sorted using Fluorescence-activated cell sorting (FACS) and the...easily determined by comparing cells expressing only eGFP expression to cells expressing eGFP and mCherry...expressed in cells. mCherry is constitutively expressed to identify successfully transfected cells. C-to-T ...eGFP and mCherry positive cells by the number of only mCherry positive cells (Martin et al., 2019). ...TREE) to purify single base edited cells without the need for single cell isolation and downstream sequencing...fluorescence and cell sorting using FACS which could limit their use across different cell types and systems...sgRNA targeting the GFPACG to the cells. As expected, only the cells that contained the cytidine base ...

...engineered cells in therapy. The lungs are difficult, but other tissues like the retina have cell-based therapies...Chapter 9: Using stem cells as in-vitro models of disease. How Darrell is using stem cells from patients to...sharing reagents, cell lines, data, and knowledge without exclusivity. Chapter 11: The Stem Cell Bank (CReM ...the lungs, and, among other projects, using stem cells to develop in vitro models for lung disease. Darrell...what drives him as a praticing physician and stem cell researcher. Darrell's ultimate goal - to develop...means to treat disease through tissue repair, stem cells, gene therapy, and tissue engineering. How the multidisciplinary...projects - differentiating surfactant producing cells that can be used to better understand the biology...

...vectors designed to express nucleases in different cell types. Zinc finger arrays can be transferred in-...vectors designed to express nucleases in different cell types. Zinc finger arrays created using ZFC reagents...nuclease domains: pST1374 (for expression in mammalian cells or zebrafish) pDW1775 (for expression in plants)... Miller et al., Nat Biotech 2007 ) in mammalian cells or zebrafish (can be used as a substitute for pMLM335... Miller et al., Nat Biotech 2007 ) in mammalian cells or zebrafish (can be used as a substitute for pMLM336... Miller et al., Nat Biotech 2007 ) in mammalian cells or zebrafish). pMLM802 (for expression of a ZFN ... Miller et al., Nat Biotech 2007 ) in mammalian cells or zebrafish). pMG10 (a '“2-in-1” ZFN cassette for...

...transfected cells, we count the number of tag-positive cells (tag ICC) as a percentage of total cells in the...Deliver csRNP into cells: Prepare cells for transfection, add csRNP and ssODN to the cell suspension, transfect...csRNP plus ssODN, is then transfected into stem cells. Cells are allowed to recover for 2-5 days and then...efficiently these components are delivered into cells; for each cell type, the transfection/delivery step needs...Proteins - the workhorses of the cell – never work alone in the cellular milieu. It is, therefore, critical...DNA) to perform diverse biochemical tasks in the cell. One of the most popular approaches to study protein...with its associated protein complexes - from the cell-lysate using antibodies against the target protein...

...unstable, meaning that the cells must continually produce antitoxin to avoid cell death. In the case of plasmids...addiction systems” require dividing cells to retain plasmids - if a cell does not inherit plasmid due to ...will be degraded leaving the cell to be killed by the more stable toxin. Cells only survive if they retain...10% of cells while plasmids containing the plasmid addiction system were retained in 75% of cells. They...designated this fragment as the ccdB region (for “coupled cell division”). The encoded toxin-antitoxin system consists...cleaved complex. This results in DNA breakage and cell death. Conversely, CcdA binds to and blocks the ... replaced by the gene of interest. As a result, cells that retain the ccdB gene die off and a higher percentage...

...untreated cells—but can we be certain? Do we know whether the plasmid delivered into the cells successfully...amount of DNA transfected into the cells and the ability of the cells to express the protein.   In analysis...Gene X in backbone Y) was transfected into human cells. From this result one could simply conclude that...didn’t work, as the expression level of Gene X in cells treated with Plasmid A is similar to the expression...or transfection process) may have on the target cells. One transfection control is an empty vector control... itself has any cytotoxic effects on the target cells. Another type of transfection control is an internal...plasmid or transfected into a separate well of your cells. Regardless, the amount of reporter protein activity...

...lines expressing GFP in specific cell types to do more than just label cells. If GFP could be co-opted to ...reporter output was observed in their 293 cell culture system. In cells cotransfected with GFP and the GBPs,...expression was induced in ~76% of GFP+ cells, and 100% of DsRed+ cells also expressed GFP. Using AAV constructs...fluorescent proteins as scaffolds for cell-specific gene manipulation.” Cell 154(4) (2013): 928-939. PubMed ...wanted to express a variety of proteins in single cell types in the mouse, but knew it would take years...could then selectively manipulate only GFP-labeled cells. Once Tang and Cepko found a description of GFP-...heavy chain, and they are small and stable inside cells. Nanobodies binding GFP were designed in 2009. Subsequent...