...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...

...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...

...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...

...the producer cells and purification protocols often describe a process that begins with cell lysis to release... steps to separate cell culture supernatant containing exo-AVVs from producer cells, and then to enrich...effectively target less accessible cells, such as brain tumor stromal cells in a mouse model of glioblastoma...particles is able to infect more cells (as demonstrated in different cellular environments such as retina ...Exosomal vesicles secreted from producer cells also contain cellular macromolecules (e.g. proteins, nucleic...Standard AAV vectors are generated with a producer cell line like the widely used 293T. During vector production... to be spontaneously released from the producer cells into the supernatant during the production phase...

...improved cell-free system to examine biosynthetic pathways A new tool for enriching ABE-targeted cells The ...interest, and cell extract.    Graphical Abstract: Schematic representation of cell-free system,...successfully reconstituted in this cell-free system. In summary, this cell free system allows for a relatively...multiplexed and can be used in human pluripotent stem cells, a cell type that has been difficult to modify in the... Corner New ready-to-use viral preps   Improved cell-free transcription/translation system to examine...biosynthetic pathways  By Gabrielle Clouse Recently, cell-free transcription/translation systems (also referred....0c00581   New tool for enrichment of ABE-targeted cells By Ellie Adams Single base modifications through...

...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...ability for more robust editing in human cell lines and primary cells makes them a great addition to the base...these base changes, especially in primary human cells. Now, scientists at Beam Therapeutics have found...

...cancerous cells Luckily, there are several safety mechanisms in place to protect us from rogue cells when ...process of programmed cell death, or apoptosis, is one such mechanism. When a cell or its genetic material... directly inhibit the ability of a cell to undergo programmed cell death while simultaneously stimulating...eliminate every cancerous cell. Even so, to pose the greatest threat, cancerous cells still need to successfully...universally-dividing behavior of cancer cells, attacking the cellular machinery necessary for division. This...proteins that, when expressed, normally encourage cell survival, growth and proliferation, while tumor ... suppressors perform the opposite task, keeping cells from dividing out of control. One may compare the...

...with live host cells Now you can proceed with incubating this membrane with your cells of interest and...membranes well with PBST to remove unbound cells. To detect bound cells, simply lay each membrane on an agar... cloned into E. coli cells, isolated colonies will be lifted onto nitrocellulose membranes and exposed... proteins directly onto the nitrocellulose for subsequent probing for cell-binding activity.  First, cut...E. coli or Salmonella, or any cells that bind strongly to nitrocellulose, over-incubation of plates can...directly onto nitrocellulose and carry out the steps from blocking the membrane to probing with cells in order...structure(s) a given phage will recognize on a host cell. To make matters even more interesting, much of...

...demonstrated its use to probe cell mechanics, vesicle transport, and cell division, but given the long ... expressed in a monolayer of endothelial cells, showing cell protrusions and overlaps (arrows) after blue...2016). Rho GTPase signalling in cell migration. Current Opinion in Cell Biology, 36, 103–112. https://doi.org...Optogenetics gives you the power to control cells or organisms with the flip of a switch. You may be ...optogenetic tools to control the cytoskeleton and cell membrane dynamics. First, a quick refresher.  The...important components of the cytoskeleton. They provide cells with physical structure, separate chromosomes during...strategies are available to disrupt microtubules in cells, they have various disadvantages and limitations...

...at bioRxiv. AAV2 interacts with the packaging cell's cell membrane When packaging adeno-associated viral...of the packaging cells (Vandenberghe, 2010). Rather than being released into the cell culture supernatant... particles associate with the cell membrane and are taken into the cell via endocytosis. In our study,...AAV2 viral particles by our packaging cells by treating the cells with the endocytosis inhibitor sucrose...target cells is blocked following sucrose treatment. Moreover, several reports have shown that cells treated... yields approximately 1.8-fold without affecting cell viability. This effect appears to be serotype specific...difference between untreated and sucrose treated cells. Although we were unable to deduce the mechanism...

...recombined cells. Recombination is a rare event, so you must select as opposed to screen for cells where recombination...Correctly recombined cells will not contain the negative selection marker, but cells with random recombination...modified version of the targeted sequence to the cell which will recombine the template with the endogenous... used to inactivate specific genes in mammalian cells. Despite the popularity of CRISPR-based knockout...targeting construct. For recombination to occur in a cell, a minimum of 2 kb of sequence homology is required...antibiotic resistance gene. To avoid selecting for cells in which the construct has randomly integrated into...outside one of the homology arms. When we select cells later, we will first perform a positive selection...

...efficient gene deletion in B cells, T cells, macrophages, and dendritic cells, with no deleterious effects...to HEK293FT and HeLa cell lines. When the miRNA activity profile for a given cell type is already known...or ZFN to edit immune cells poses challenges such as the inhibition of immune cell differentiation. Furthermore...immune cell activity. However, a CRISPR-based tool for in vivo functional analysis of immune cells has been...construct in bone marrow stem cells from Cas9-expressing mice, and used the cells to reconstitute the immune..., these reporters can help define the different cells present in a mixed population, or identify differentiation...turning the single-input reporters into multi-input cell classifiers in one assembly step. Find the miRNA...

...dividing cells only, so is not suitable for infection of quiescent cells or infrequently cycling cells but ...non-dividing cells, making it conducive to targeting all cell types. Altogether, AAV has many excellent features...DNA copies of their genome upon infecting a host cell. This viral DNA (also called proviral DNA) is then...and permanently integrate a genetic cargo into a cell. These viruses have a cargo capacity of ~ 8 kb, ...needs, and can transduce (infect) a wide range of cell types with a high efficiency. However, gamma-retrovirus...but is fine for experiments with immortalized cell line cultures. Lentivirus Lentivirus is a type of retrovirus...lentivirus can infect both dividing and non-dividing cells. Lenti particles enter the nucleus through nuclear...

...and yeast cells are cost-effective and faster-growing than mammalian cells, but mammalian cells can perform...your cells with your plasmid(s), and then wait 7-14 days (in mammalian cells) to collect the cell culture...production system, cell lines typically used include Human Embryonic Kidney (HEK) cells and Chinese Hamster...recombinant antibodies with some molecular biology and cell culture experience. Let’s go over the basics of ... place in bacterial, yeast, plant, or mammalian cells. Each of these systems has their advantages and ...post-translational modifications that bacterial and most yeast cells cannot. Your choice of production system may depend...Hamster Ovary (CHO) cells. (L'Abbé et al., 2018).  Recombinant antibodies can be produced through a single...

...transduction of cells, resulting in cell population mutated by the viral pooled library. These cells can then...transduce your cells.  Titering After generating your lentivirus, you’ll want to infect your cells with the ...of a cell being infected by only one viral particle, but it also increases the chances that a cell doesn...adequately represented in the cell population, you’ll need to infect many more cells than the number of plasmids...appropriate packaging and envelope plasmids into HEK cells allows production of lentivirus. Purification of...small aliquot is not enough to transfect producer cells, so your first step will be to amplify the library...approach for producing lentivirus in Lenti-X 293T cells using a polyethyenimine transfection protocol. I...

...Cas9 in Intestinal Stem Cell Organoids of Cystic Fibrosis Patients." Cell Stem Cell. 13, 653-658 (2013). ...patient’s own cells that have been modified ex vivo, previously edited healthy donor cells can be delivered...Genetic Disease in Mouse Via Use of CRISPR/Cas9." Cell Stem Cell. 13, 659-662 (2013). Gerald Schwank et al. ...on editing animal models of disease and cultured cells. For example, in 2013, Jinsong Li and colleagues...applied CRISPR to disease correction in adult stem cells isolated from two patients with cystic fibrosis ...delivery directly to the patient. Patient-derived cells can alternatively be harvested, edited ex vivo, ...therapies involve editing healthy donor-derived cells and delivering them to the patient.   Current...

...copy number in the cell is high, but allows replication when copy number in the cell falls too low (Novcik...system to partition themselves to each daughter cell during cell division. They don’t simply rely on chance...eukaryotic cells). The partition NTPase is recruited and “walks” each plasmid to a separate daughter cell. When... daughter cell, instead of partitioning each type of plasmid into a separate daughter cells (Figure 2)...from the cell with the same probability, or asymmetrical, where one plasmid lost from the cell at a higher...different plasmids to be maintained in one bacterial cell. In this Plasmids 101 post, we’ll cover why this...incompatibility occurs when multiple plasmids within one cell have the same replicon and/or partitioning system...