...produced using complex mixtures derived from cells or using live cells. There are several types of expression...consists of transfecting cells with your DNA template of choice and allowing these cells to transcribe, translate...demanding cell culture conditions compared to other systems. Insect expression systems Insect cells can also...coli or yeast cells. The only problem with baculovirus systems is that the infected host cell is eventually...eventually lysed. Cell lysis halts protein production, but there are non-lytic insect cell expression systems...Sf21, Hi-5 cells)  that allow for continuous expression of genes integrated into the insect cell genome. ...that is then transformed into a bacterial cell. Transformed cells propagate, are induced to produce your ...

...Love your cells and they'll love you back A successful lentiviral infection begins with your cells, both ...both your packaging cells and your target cells. Poor tissue culture practices can lead to low titers and... promote cell growth. Overgrown cultures stimulate quiescence, the resting phase of the cell cycle; quiescent...quiescent cells do not take up nucleic acid or express transgenes as well as actively dividing cells resulting...reaching 108 cells per ml before the media becomes cloudy. Mycoplasma compete with host cells for nutrients... your producer cells with fresh medium. Daily medium exchanges help alleviate cellular stresses associated...consumption due to the high cell density of the culture. In addition to benefitting your cells, medium exchanges...

...embryonic kidney (HEK) cells originated from? Or why they are such a commonly used cell culture model? In ...isolating the cell line all the way to modern applications. What the HEK is it?   HEK cells were first ...inconsistent with kidney cells (Shaw et al.). Since then, transcriptome studies of the cell line have determined...for?   As one of the older cell lines in the research community, many cellular research tools have been ...any unwanted material (HEK cells, etc.) and are ready to use on another cell or organism. This pipeline...where HEK cells may be particularly useful due to their intrinsic properties. HEK-293 cells are tumorigenic...researchers using the cell line. What the HEK is wrong with it?   All cultured human cell lines are susceptible...

...producing cells. After FACS, return the cells to doxycycline-free media and expand the cells. Use a subset...white inclusions in cells with GVs via phase contrast microscopy, GVs visible in cells via TEM, and purified... heterogeneity required single cell screening to isolate a clonal cell line capable of producing ultrasound...expression homogeneity, as the cells could be batch-sorted to isolate a polyclonal cell line with strong gas vesicle...transient transfection in rtTA+ cell lines (such as HEK293-TETOn cells), or they can also be integrated...PiggyBac transposase. In rtTA-cell lines, it is better to first engineer the cell line with rtTA, then integrate...own mARGAna cell line. (c) Z-projected phase contrast images of GV expressing MDA-MB-231 cells (top, scalebar...

...determine cell cycle length by examining the ratio of blue to red cells. Cells undergoing rapid cell cycles...engineered C. elegans cell lines to express wrmScarlet1-10 unattached to any other cellular protein. They used...’s difficult to observe the cell cycle in vivo, particularly if the cells are migratory or in deep tissues... BaF3 cells were transduced with H2B-FT virus and grown in varying concentrations of IL-3. Cells grown...proportion of blue fluorescence, indicating cell proliferation. BaF3 cells maintained in media without IL-3 show...H2B-FT consistently predicted the cell cycle speed across these different cell types. In one example, H2B-FT...expressed in murine B cells (BaF3), which depend on IL-3 to proliferate. The cells cultured with a low ...

...infected cells, the other using wild-type Cas9 to remove the HIV-1 genome from infected cells. Read on...1 reservoirs HIV-1 infects cells in the immune system, notably CD4+ T-cells, and eventually leads to acquired...percentage of cells with activated HIV-1, measured via HIV-1 promoter-driven GFP expression. In cell lines that... genome from every infected cell and 2. prevent reinfection of those cells. Kaminski et al. designed gRNAs...Kaminski et al. observed no negative effects on cell viability, cell cycle progression, or apoptosis with Cas9...in HIV-1 infected T-cells to see if they could be rescued by CRISPR. CD4+ T-cells were isolated from healthy...1 is limited to a subset of T-cells, but it is unclear if enough T-cells could be reached to ablate a ...

...on or in these cells: antibodies can bind to proteins on the cell surface, or the cell membrane can be...antibodies are produced by B cells. They bind to proteins on the surface of extracellular pathogens such as parasites...fusing B cells that have been extracted from animals injected with protein antigen to myeloma cells. The ... measurement of protein expression in cell lysates, whole cells, or tissue samples. Many experiments that...heterogeneous mixture - for example, cell lysate or media from cultured cells. ELISA can be used to quantitatively...same cell simultaneously as well as compare between samples. Learn more about flow cytometry. Cell and ... (immunohistochemistry) or cells (immunocytochemistry). This could be cells or tissue slices mounted to...

... HEK293FT cells expressing a single gRNA targeting a single gene. The gRNA expressing cells were then ...plasmid to generate a population of mutant cells. The mutant cells were treated with WNV at a dose and duration...regulate the ability of WNV to induce cell death in infected cells (SEL1L, UBE2J1, EMC3, EMC2, DERL2, UBE2G2...small GTPases involved in cell signaling pathways that control many cellular processes such as differentiation...therefore varies with cellular location: it is both red and green through much of the cell, but turns more ...AID-tagging system to essential genes in human cells and mouse ES cells because it was difficult to fuse the AID...-to-study genes in hard-to-study cell types. Natsume T, et al. Cell Rep. 2016. PubMed PMID: 27052166 ...

...infecting most cells, but not all. For example, LDLR expression in hematopoietic stem cells, T cells, and B cells... host cell. Some of these envelope-receptor pairings are broad, allowing delivery into many cell types...delivery to only a few cell types.  If you’re looking for more control in the cell types your lentiviral...protein causes cells that it infects to fuse together and form syncytias that cause the cells to die. Cytotoxicity...Cytotoxicity can also be cell type dependent: if a cell has few receptors for a specific envelope protein...lipoprotein receptors (LDLRs) on a cell surface to endocytose into the cell. The reason VSV-G is so commonly... cells is low. Infection of these cells with VSV-G pseudotyped virus requires the upregulation of LDLR...

...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...Proteins - the workhorses of the cell – never work alone in the cellular milieu. It is, therefore, critical...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...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...

...human embryonic stem cells. Transduced stem cells were differentiated and single cell RNA-seq was performed...transduced human embryonic stem cells with the viral TF library, with each cell receiving only 1 TF expression...construct. Cells were cultured following a differentiation protocol before performing single-cell RNA sequencing... 206 cells per TF ORF. From this large sequencing endeavor, a map of possible TF-induced cellular states...differentiation.   Fig. 2: Single cell RNA-seq clustering of cells transduced with the TF pooled library... clusters of individual cells along with color coded nominated reference cell types.  (Adapted from Joung...delivered to each cell, future applications could deliver multiple TFs to each cell, allowing for combinatorial...

...about RapID! EXCELL: Proximity labeling for the detection of cell-cell interactions EXCELL is an enzyme-mediated...enzyme-mediated proximity cell labeling strategy to detect cell-cell interactions in vivo (Ge et al., 2019...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...peptide (LPETG). In EXCELL, the mgSrtA variant can be displayed on the surface of a cell of interest, where...-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...

... your cell type of interest, while miRNA target sites can be used to prevent expression in cell types ...endothelial cells in rodent CNS AAV-PHP.V1 shows higher transduction in CNS endothelial cells (60%) and ...endothelial cells in the retina and spinal cord (Krolak et al., 2022). Systemic capsids for broad cell type ...mouse to primate neocortex. Cell Reports, 34(13). https://doi.org/10.1016/j.celrep.2021.108754 Ravindra Kumar...delivery across species. Cell, 184(19), 4919-4938.e22. https://doi.org/10.1016/j.cell.2021.08.028 Vormstein-Schneider...capsid, (b) cargo, and (c) delivery method determine cell type- or region-specific transgene expression. (...include variants with broad CNS or PNS transduction, cell type specificity, or retrograde transport in neurons...

... 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...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...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...

...transgenic cell lines? One of the major uses of the piggyBac™ system is to create stable transgenic cell lines...efficiency is likely to be cell line-dependent, and should be tested in the cell line of interest. Importantly...If the cell’s chromatin structure undergoes significant rearrangement (such as when a stem cell initiates...manipulations in animal models or cells lines, including stem cells, without leaving behind any accessory...applications, including making transgenic cell lines and genetically-modified cell lines. Furthermore, the use of...molecular and cellular biology and brings 10 years of research experience with stem cells to Transposagen's... in Human Pluripotent Stem Cells: Choosing the Correct Path.,” J. stem cell Regen. Biol., vol. 1, no. ...

... into cells in the lab. The introduction of foreign DNA or RNA into bacteria or eukaryotic cells is a ...application of an electrical charge to cells, to increase cell membrane permeability and thus transformation...introduce foreign DNA into eukaryotic cells, like mammalian cell lines. This can be done with lentiviral...the genome and stable cell lines, where foreign DNA is incorporated into the cell’s genome and is thus passed... pokes holes in the cell membrane so DNA can be introduced directly into the cell. Microinjection requires...multiple ways foreign DNA can be introduced into cells including transformation, transduction, conjugation...genetic material from the environment by bacterial cells. In nature, this genetic material often comes from...

... the single cell level), are separated by, for example, cell sorting. The separated cells are subsequently...more than 8x107 successfully targeted cells. Depending on the cells used and the readout, this could be ...My group works on immune cells in different contexts. Most of the immune cells we study are migratory, ...CRISPR modified cells are transferred into an animal and the behavior of the transferred cells is followed...with an easily measureable cellular phenotype (e.g. survival of a cancer cell as a cytotoxic drug is added...genes essential for West-Nile-virus-induced cell death." Cell reports 12.4 (2015): 673-683. PubMed PMID:...With these screens, the investigator generates a cell population where all genes are knocked out at the...

...model systems: either human cells or mice. In a recent issue of Cell Stem Cell, Musunuru, Chad Cowan and...done in stem cells, we can differentiate those mutant and wild type stem cells into any cell type relevant...system for generating human stem cell-based disease models." Cell Stem Cell. 12, 238-251 (7 February 2013...homology-directed repair by flooding the cell with a mutant template and fooling the cell into using that template for...mutations influence the disease process. In the Cell Stem Cell paper, for example, we generated hepatocytes...generating the mutant cell lines that one needs to do disease modeling in stem cells. Addgene: Tell me about...Kiran Musunuru's lab in Harvard's Department of Stem Cell and Regenerative Biology is to understand the basis...

...molecular level, at the cellular level there is a universal theme - uncontrolled cellular proliferation. We ...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...promising results in the laboratory, and Addgene is accelerating this research by helping to make new cancer ...

... target to specific subcellular regions, laser ablation can damage other cellular structures, and genetic...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...