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... a big issue for cells, but it may confound research results or even kill your cells if your gene of interest...necessary to replicate plasmids and make sure the cells maintains it. Here, we'll cover the promoter, which... Because transcription machinery differs between cell types or organisms, promoters must be similarly ...variable. Bacterial promoters only work in prokaryotic cells and typically only in the same or closely related... were derived. Similarly, the various eukaryotic cell types (mammalian, yeast, plants, etc) require unique...complex, having fewer parts than those in eukaryotic cells. Some promoters are constitutively active and on... at will with a chemical, heat, or light. In the cell, promoters themselves are controlled by still other...

...dodge cellular variation observed in mammalian cells is to tag an endogenous gene. By imaging cells (or ...mammalian cells within the dish. This is caused by the stochastic nature of plasmid uptake by cells. To correct...under your experimental conditions. This is time well spent and, if you do it right, can be (part of) ...comparison of fluorescent proteins and how to make a well-informed decision on the best fluorescent protein...strongly depends on both the biological system as well as the imaging method, we recommend basing your ...intended application. The three key properties for live cell imaging that will be treated in this series of posts...with a high intrinsic brightness that does not fold well or cannot be detected in a microscopy setup has ...

...genes known to be essential for cell growth. Together, this excellent work highlights the importance of...fluorescence-activated cell sorting (FACS). This is not possible in plants outside of limited, single cell protoplast...exquisitely and robustly self-organize at the cellular and multicellular scale.     References Bezanilla M, Perroud...Generation. Plant Cell 24:1478–1493 . https://doi.org/10.1105/tpc.112.098509 Smits AH, Ziebell F, Joberty G...can be found at Addgene that could serve as an excellent start (#22988, #137884). Both vectors allow the...2018) and the model bryophyte Physcomitrium (Physcomitrella) patens (Bezanilla et al., 2005; Nakaoka et...visually identified subpopulation. For traditional cell culture, enriching your active RNAi subpopulation...

... into AAV particles for delivery into mammalian cells, especially when fused to effector proteins for ...size of the Cas9 or Cas12a based systems, making it well suited for AAV packaging. Most importantly, this..., base editing, and genome editing in mammalian cells. The “V4” version (named CasMINI) had the highest...an active nuclease.  Read the paper in Molecular Cell Find the CasMINI plasmids Efficient CRISPR editing...activity and decrease the gRNA size by intensively remodelling the gRNA scaffold through five rounds of gRNA...effective in genome editing in both bacteria and human cells using various delivery methods, including plasmid...plasmids   Selective endogenous encapsidation for cellular delivery of RNAs By Mike Lacy The retroviral-like...

...Purification Process with pCri Cell-free protein expression kit Cell-free expression systems have been...SITS to create the pCell-Free vectors, Gateway-compatible backbones that enable cell-free expression of...testing and comparison of different cell-free expression systems; the Cell-free expression test kit represents...: FX Cloning Kit BIOFAB Plasmid Set pCri System Cell Free Protein Expression Kit FREQ Seq PUF Assembly...facilitate gene expression in bacteria, yeast, mammalian cells, insects, and even Xenopus oocytes. The expression...combinations of protein tags (His, Avi, and GFP) as well as protease sites to facilitate protein purification...of proteins in both prokaryotic and eukaryotic cell extracts (Gagoski et al 2015). He has also constructed...

... defined population of cells (e.g., a neuron subtype) and specific subcellular compartments for measurements...we will explore some of the most commonly used as well as newly developed calcium sensors. Selecting a...calcium concentrations and transients in living cells and organisms. Unlike chemical indicators, these...Localization The calcium concentrations in different subcellular compartments can vary greatly. Additionally, ... violet light and (2) exposure to increased intracellular calcium. It has been applied to mark active ...neuron-specific expression (hSyn promoter) and pan-mammalian cell expression (CAG promoter). An improved version named...neuron-specific, astrocyte-specific (GCaMP6 only) or mammalian cell expression, and compatible with constitutive or ...

...can actually infect cells. These assays typically involve infecting a target cell line with your virus...distinguish between a cell that has had one viral particle integrated into its genome and a cell that has had ...arrived; you’ve painstakingly cared for your packaging cell line, prepped your DNA, transfected and harvested...move ahead with your infection and make your stable cell line. While we’ve all experienced the pressure to...Read on for an overview of the titering options as well as the benefits and drawbacks of different methods...viral copies that have integrated into the target cell’s genome. Physical titer The two most common assays...functional titer uses FACS to count the number of target cells that are positive for vector encoded transgene expression...

...reagents and keep the cells happy. Harvesting virus Once you have transfected your cells, wait 2–5 days and...however, you’ve collected the producer cells, you’ll need to lyse the cells to let the virions out. We recommend...particles that can transduce cells, which can vary based on target cells and can be altered by freeze/...several facilities nationwide where you can obtain excellent quality, high titer rAAV (including Addgene!),...come into contact with the rAAV and apply common cell culture practices diligently throughout the production...capsid genes.  In this helper-free system, HEK293T cells, expressing Ad E1A and E1B, can be transfected with...and then collect the supernatant and/or producer cells for further processing. What component(s) you need...

...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...

...Stability improvement of antibodies for extracellular and intracellular applications: CDR grafting to stable...systems, and often do not fold properly once inside cells.  Developing the HA frankenbody To combat the issues... detects the widely used HA epitope in diverse cellular environments. They called this probe the HA frankenbody...previously proven to fold and function in living cells. To make these scaffolds specific to different targets...probe structure and stability from the scaffold as well as specificity towards a HA epitope from the CDRs...the tagged protein upon probe binding in living cells.  In their proof of concept experiment the Stasevich...mCherry, 4xHA tagged histone protein (H2B) in U2OS cells. They then showed that the HA frankenbody probe ...

...agent than BME and has a less… distinctive smell. BME smells like rotting eggs but has a longer shelf life...expensive Nitrocellulose or low-fluorescence PVDF membranes Increased fragility (nitrocellulose) or increased...Both polyclonal and monoclonal antibodies can work well for a western blot. Polyclonal antibodies, which...optimization and likely higher background noise as well, and multiplexing is challenging and limited. Direct... mechanical method before your enzyme lysis. For cell cultures, enzyme lysis is typically sufficient.  ...When choosing your lysis buffer, consider the subcellular location of your protein. Proteins located in...proteins easily available in the cytoplasm or whole-cell lysate. A harsher, SDS-containing lysis buffer, ...

...purify DNA, cellulose also efficiently binds, or at least entraps nucleic acids. The Botella lab took advantage...broken out of cells with a solution of salts, detergent, and ball bearings. Then a cellulose dipstick is... kits and typically follow the same principles: cells are lysed, RNAs and proteins are removed to leave...four methods for DNA purification without a kit as well as one way to reuse silica columns from DNA purification...glass. After binding DNA, the glass particles are pelleted by centrifugation and washed before DNA is eluted... could be digested with restriction enzymes.  Cellulose nucleic acid purification Starting material: plant... plant, microbe, blood, or mammalian cells or tissue Product: total nucleic acids While glass or silica...

...protein localization, as well as cellular processes like gene expression and cell migration (Sladitschek... MXS-chaining were used to label subcellular structures in live cells. Four individual expression constructs...construct was then introduced to HeLa cells (Figure 1). The resulting HeLa cells show robust labeling, with strong...High-throughput cloning, in a nutshell, is the systematic combination of different genetic sequences ...imaging or flow cytometry applications in mammalian cells. The modules used in this method include fluorescent...components, one can generate constructs to visualize cell cycle dynamics, titrate inducible transgene expression...detection of each fluorophore at the respective subcellular structure (Figure 1). Here, MXS Chaining gave...

... for stem cells, or metastasized tumor cells, or alzheimers-affected neurons, or [your cell line of interest...critical to stem cell survival. Knowing which genes influence the survival of our cell culture is the foundation...minimal side-effects on healthy cells and maximal effect on unhealthy cells. Until recently, the human genome...ENCODE project, find enhancers in embryonic stem cells, and conclude with a targeted CRISPR screen that...essential transcription factor in embryonic stem cells (ESCs). Your ChIP-seq finds just over 13,000 significant...We start by infecting millions of embryonic stem cells (ESCs) with a lentiviral vector, an attenuated retrovirus...The result is a heterogenous population of stem cells, usually housed in a single flask. Through a bit...

...Cas9 in Intestinal Stem Cell Organoids of Cystic Fibrosis Patients." Cell Stem Cell. 13, 653-658 (5 December...CRISPR in adult stem cells has huge potential, because the genomes of adult stem cells in organoid cultures...Genetic Disease in Mouse Via Use of CRISPR/Cas9." Cell Stem Cell. 13, 659-662 (5 December 2013). 8. Gerald Schwank...connected to the early onset of Alzheimer’s disease, as well as those that play into late onset AD (5).     ...homologous recombination in cultured intestinal stem cells of patients with cystic fibrosis (8).   The difference...applied CRISPR to disease correction in adult stem cells isolated from two patients with cystic fibrosis....is a big advantage over induced pluripotent stem cells, which need to go through a phase of crisis where...

...By taking advantage of repair pathways within the cell — in some cases, the same repair pathways that were...adenosine (A) to inosine (I, treated as G by the cell). Surprisingly, the team noticed that this editor...an inhibitor of base excision repair (BER), the cellular process that catches mutated U’s in DNA and ideally...cytosine editing between bacteria and mammalian cells Researchers in the Xueli Zhang lab were making similar...2021). Their GBE produced C → G edits in HEK293T cells with over 50% efficiency in 23 out of 30 target ...follow the same initial mechanism as in mammalian cells — by excising the freshly edited U bases — but E...the base editor. Black arrows indicate natural cellular processes. Created with BioRender.com. Beyond...

...thought to activate programmed cell death or a dormant state for bacterial cells that have been infected with...collateral RNA cleavage response is well described in bacterial cells, LwaCas13a does not display nonspecific...eukaryotic cells. They arrived at this finding through analyses of global RNA expression, cell stress, and...they identified 53 potential candidate genes that fell into 3 categories based on the architecture of the...target RNAs, although its usefulness in bacterial cells will likely be limited by the propensity of Cas13a...pool of RNAs) or studying RNA processing in live cells. Indeed, groups have utilized dCas13 fusion proteins...genotype human DNA, identify tumor mutations within cell-free genomic DNA, and detect COVID-19 (Joung, Ladha...

...isolating the B cells from an immunized animal, those B cells are fused to myeloma cells at a 1:1 ratio...fusion between an antibody-producing B cell and a myeloma cell (National Research Council (US) Committee...immortal cell population that will continue to produce the specific antibody that the original B cell produced... selects against unfused myeloma cells and antibody-producing B cells that cannot replicate, meaning that...monoclonal antibodies are derived from a single B cell clone. One common way of producing monoclonal antibodies...with BioRender.com.   The antibody-producing B cells that are used to make hybridomas come from animals...antibody-encoding genes over time as the hybridoma cells divide. These changes can alter the antibody that...

...difficult-to-transfect cell types ranging from primary to embryonic stem cells. Viral integration of a... also generally less toxic to cells than transfections, preserving cell viability. Library delivery Screening...Viral applications Viruses work by infecting a host cell (the target of viral infection) and delivering a...to be anything you want delivered to your target cells. This delivered DNA/RNA can then be either permanently...permanently integrated into the genome of the target cell or transiently expressed. It’s important to note...This vector can express your construct in target cells transiently or constitutively depending on the virus...transcription factors, barcoded libraries to trace cell lineage, and so much more! Libraries allow you to...

... immunology, cancer-immune cell interactions, cancer immunotherapy, as well as new approaches to reprogramming... fields of Cancer, Inflammation and Immunity, as well as various combinations thereof (so called “Immuno-Oncology...cancers. It was inspiring to participate in the 2017 Cell Symposium on Cancer, Inflammation and Immunity and...immunosurveillance. The presented study analysed a model of hepatocellular carcinoma, a cancer which can develop from ...example the upregulation of the immunosuppressive cell surface receptor PD-1. This immunosuppressive environment...follow the interactions between tumor and immune cells in real time. Addgene depositor Professor Max Krummel...effect of PD-1 blockade.“ (Ref 4). PD-1 helps tumor cells evade the immune system - COX inhibitors like aspirin...