... therapy.” Journal of cellular physiology.  https://doi.org/10.1002/jcp.30064 2. Barman et al. 2020. ...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...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...

...improve the brightness of previous Nano-lanterns by 2-4 fold and permit five-color fluorescent imaging! ...have become indispensable tools for molecular and cell biologists over the last two decades, but their ...light. Long-term light exposure can also lead to cellular phototoxicity, and experimental success can be...quantum yield, and when transiently expressed in cells, generates much less signal than comparable fluorescent...three luminescent proteins can be used together in cells. As Nano-lanterns are not subject to photobleaching...phototoxicity, they successfully imaged multiple subcellular compartments over several minutes using Nano-...concentrations of the compound can be added to the media. Cellular esterase then converts diacetyl coelenterazine-h...

...to visualize multiprotein complexes.  Fig. 2. Multiplex immunolabeling with subclass-switched recombinant...cryopreserved hybridomas, immortalized cells created by fusing an activated B cell that produces the antibody of...of interest with a myeloma cell. This RNA was then used to generate cDNA, which  was used as a template...of hybridoma generated mAbs is that if the frozen cell line becomes non-recoverable, the stock of the mAb...hybridoma stocks, without having to grow up the cells. This RNA could then be used to generate functional...immunofluorescence-immunocytochemistry (IF-ICC.) This method was chosen because cells transfected with a plasmid to express the target... could be screened in a high-throughput manner. Cells were labeled with both a hybridoma generated mAb...

...work with Grows extremely fast (doubling time of ~ 2 hours) Amenable to genetic manipulation The first...path.   Yeast as a model organism Yeast are single cell, eukaryotic organisms formally classified as fungi...and S. cerevisiae (budding). Yeast make for an excellent model organism for a few reasons: Affordable...similarity to the ancient yeast strains and metazoan cells.   Applications for K. phaffii There’s a number ...protein bang for your media buck - it grows at higher cell densities than most of its counterparts, yielding...oxidative reactions. They are found in all eukaryotic cells and peroxisomes are where methanol metabolism occurs...in methylotrophic organisms. K. phaffii is an excellent model for peroxisome studies since their peroxisomes...

...labeling of different cell types within a sample to expected labeling (Figure 2). The panel of antibodies...the antibody in the same tube.     Figure 2: Schematic showing the use of an orthogonal approach...levels in your cell lines, you can consider a cell treatment approach. If there is a cell treatment (e....select a candidate cell line (e.g. DepMap) – you’ll need to ensure that the parental cell lines of your chosen...the parental cell line, but not in the iso-genic knockout control. This can involve cell surface labeling...selectivity for cell surface labeling may become less selective if used in intracellular labeling because...because the intracellular compartment may contain cross-reacting antigens not present on the cell surface....

...retransduce a model organism such as mice, e.g. first at 2 months, then at 4 months? Or do antibodies become ...co-delivering different AAV particles into the same cells in vivo? A: This will be highly dependent on vector...particularly one that can target the epithelial cells in the blood-brain barrier?   A: For liver, we recommend...worth investigating for both Q6 & Q7.For epithelial cells of the blood brain barrier we would currently recommend...and to all our participants for their time and excellent questions.    Additional Resources on the Addgene...

...axon terminals to the soma (retrograde tracer) (Fig. 2). Viruses as neuronal scalpels Viruses can be injected...they either trace downstream from the soma (neuron cell body) toward the axon (anterograde tracer) or upstream...particles require receptor-mediated entry into the cell. This means that not all neurons and pathways will...chemogenetic vector directly into the region where the cell bodies of the projection neurons reside.   Impact...

...two options to begin your plasmid deposit. Figure 2: Choose to submit plasmid(s) online or by using a ...format. Figure 6: Add sequence, maps, and files. Step 2: Gene and Insert *Note: This page will not appear ...for a lentiviral vector to be used in mammalian cells, check Lentiviral and Mammalian Expression). This...

...containing complementary polynucleotide chains. J Virol 9(2): 394-396. Lecomte E, Tournaire B, Cogné B, Dupont... small amount of spurious DNA from the packaging cell line, helper and packaging plasmids. We decided ...

...extraction protocol for more details.   Figure 2: An uncut plasmid is expected to form 3 bands: nicked...efficient genome editing in Escherichia coli. Microb Cell Fact 19. https://doi.org/10.1186/s12934-020-01321...

...delivering an AAV construct expressing channelrhodopsin 2 (ChR2), a blue light sensitive cation channel, fused...and chemically excited a class of enteroendocrine cells within the intestines known as neuropods. They observed...

...PubMed PMID: 8491198. PubMed Central PMCID: PMC413431. 2. Bukrinsky MI, et al. “Active nuclear import of human...Non-Dividing Cells Transient Gene Expression Low Tropism for Mouse Cells and Hematopoietic Cells Low Packaging...fuses with the target cell’s membrane, allowing the viral particle access into the cell. The viral envelope... of cell the virus will target and fuse with. In nature, this determines which organisms and cells they...viruses can be engineered to be cell type specific. Once inside the host cell, the virus unleashes its cargo...wide range of cell types that are typically ‘difficult to transfect’, such as primary cells. They can also... them to easily deliver their “cargo” to target cells and organisms, and when that cargo is designed to...

... would release them into the cytosol. Fig. 2: Endogenous sources of duplexed RNA DNA:RNA hybrids...mammalian cells. Here, we will cover how these structures are generated, why it’s important for cells to sense...., Hur, S. Cellular origins of dsRNA, their recognition and consequences. Nat Rev Mol Cell Biol, 23: 286...performs almost all of its biological functions in our cells in the single strand form, but double stranded RNA...with bidirectional transcription. In an uninfected cell, dsRNA rarely circulates, but upon viral infection... Fig. 1: Exogenous sources of dsRNA Cellular sources of dsRNA Transposable elements such as...While these molecules are clearly associated with cellular damage and dysfunction, it is unclear what the...

...Gene and Cell-Based Therapies in the New Era of Personalized Medicine. Genes Dis. 2017 Jun;4(2):43-63. ...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...

... Figure 2: Isotype switching requires DNA recombination of the antibody gene locus. B cells start by ...the surface of basophils and mast cells, two types of white blood cells involved in allergic reactions. ... is important because it determines what immune cells and molecules are recruited by the antibody to help...each of these functions: Recruitment of immune cells that express receptors that recognize the Fc portion...which can initiate a cascade that helps attack extracellular pathogens.  Transportation of antibodies to ...special receptors that transport antibodies through cells and into different body compartments, such as into... during a primary immune response because all B cells begin by expressing IgM. IgM has a pentamer structure...

... vaccines, including 1) cellular responses in addition to antibody response 2) very high immunogenicity...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...

... those that also infect the liver.    Fig. 2: Summary of the generational evolution of the AAV .... Nat Cell Biol. 2022. https://doi.org/10.1038/s41556-021-00836-1 Visual barcodes for live cell clonal...with one visual barcode per cell and up to 200-plex with two barcodes per cell. The researchers validated...long sequence knock-ins Visual barcodes for live cell clonal multiplexing Improving flexibility for ... powerful approach for proteomic mapping in both cell culture and whole organisms (see our 2019 overview...reduced risks of unwanted mutations in mammalian cells. Long sequences traditionally have higher risks ...multiplexing by: Rachel Leeson Multiplexed images in live cells remains a challenge due to multiple factors, including...

... the 2 micron circle (a natural yeast plasmid) allows for 50+ copies to stably propogate per cell. The...within an E. coli cell, but vectors can be widely utilized across many different cell types and each one... the yeast cell. These elements control not only the number of plasmids found in each cell, but also whether...are eukaryotes and thus contain complex internal cell structures similar to those of plants and animals...components to allow shuttling between E. coli and yeast cells. To be useful in the lab, the vectors must contain...replication (ORI) and a means of selection in yeast cells, in addition to the bacterial ORI and antibiotic...determines plasmid copy number within the bacterial cell. Similary, the specific ORI elements included within...

...closely mimicking the future application. In figure 2 we provide an example of the comparison of the practical...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...intended application. The three key properties for live cell imaging that will be treated in this series of posts...Determining practical brightness When a dish of mammalian cells is transiently transfected with a plasmid containing...allows for a precise determination of brightness in cells, under realistic experimental conditions. By determining...al (2017) and Heppert et al (2016). In mammalian cells this would be possible with CRISPR/Cas-based genome...

...acceptable variations in DR sequences. Figure 2: comparison of Cas 9 vs. Cas 12a expression cassettes... including live-cell imaging, single-molecule tracking, immunolabeling of fixed cells, Western blotting...possibilities in both cell engineering and gene therapy.  Xiaoshu X, et al. Molecular Cell. 2021. https://doi.org...Anti-FLAG Frankenbodies for modular imaging of cellular targets Optimized genetic screening technique ...Anti-FLAG Frankenbodies for modular imaging of cellular targets by: Mike Lacy Adding to their set of anti-HA...well-folded scFv scaffold to improve its stability in cells, and use it in a variety of applications. This set...frankenbodies to amplify the signal.  Liu et al. Genes Cells. 2021. https://doi.org/10.1111/gtc.12893, Zhao et...