... 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 ...

...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...

...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...

...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...

...aspects of the work you presented may also be unclear. 2. Make the audience your primary focus When writing...as an opportunity to practice communicating.  Excellent scientists learn from advisers and other mentors...help graduate students in all STEM fields become excellent science communicators. To empower graduate students...

...higher quenching of mScarlet emission. Figure 2: Ace-mScarlet is a fusion between the voltage-sensitive...This novel tool is practical in cell and organ culture, as well as cell lineage studies. Eisenhoffer and...CreLite for cell tracing using the transgenic zebrafish model, ubi:zebrabow. In this system, cells express...fast-splicing split intein and co-infected into the cell. Intein splitting removes any exogenous sequences...split into two plasmids and co-transfected into cells for reconstitution. Image from Levy et al., 2020...red-light inducible optogenetic tool for precision in cell lineage tracing Article contributed by Erin Sanders...fluorescent proteins controlled by Cre recombination. Each cell can have multiple copies of the transgene that recombine...

...about colony PCR in this blog post.   Figure 2: The steps of colony PCR.   Sanger sequencing Sanger...activity. These cells, presumably the cell you want, cannot break down X-gal and are white. Cells that don’t...killing off a portion of the cells like in a selection. Instead, all cells survive and you will need to... the plasmid you’re looking for out of the many cells that don’t. One way to begin the search is by using...using selection strategies, where only cells that have gained or lost a specific gene survive (ex: antibiotic... plates containing X-gal allows you to identify cells that took up the vector and distinguish between ...

...PMID: 20543841. PubMed Central PMCID: PMC2920597. 2. Nathans, Daniel. "Puromycin inhibition of protein...proteins in cells. Homeostatic regulation of protein abundance is essential for normal cell function, and...one particular protein in neurons or in any other cell type. However, we cannot distinguish new from old...facilitate the visualization of specific proteins in cells. Tagged protein can easily be overexpressed from...techniques to follow translation in living single cells. However, before CRISPR-Cas9 these techniques were...interests, suspected to interact, are added to a cellular sample. Both antibodies have a short antibody ... turnover of specific endogenous proteins in any cell type. Exciting! Many thanks to our guest blogger...

...a. Plate target cells and incubate at 37°C, 5% CO 2 overnight. Day 2: b. Target cells should be approximately...plates and grow at 37°C, 5% CO 2 overnight. Day 2: b. The target cells should be approximately 80-90% ...materials D.2 Screening for inserts E. Producing Lentiviral Particles E.1 Recommended materials E.2 Protocol...Published articles H.2 Web resources I. Appendix I.1 Sequence of pLKO.1 TRC-Cloning Vector I.2 Recipes I.3 Warranty...VWR: #7177-48-2. Use at 100 μg/mL. Carbenicillin VWR: #80030-956. Use at 100 μg/mL. C.2 Annealing Oligos...oligo 5 μL Reverse oligo 5 μL 10x NEB buffer 2 35 μL ddH 2 O Incubate for 4 minutes at 95°C in a PCR machine...buffer 1 1 μL AgeI add ddH 2 O to bring to 50 μL final volume Incubate at 37°C for 2 hours. Purify with Qiaquick...

...available in the adenovirus section below!   Fig. 2 - Viral vector components of AAV Adenovirus Adenoviruses...genetic cargo, typically an expression construct, to cells/organisms. This is often cDNA, shRNA, or something...perform very similar functions to infiltrate the host cell and deposit viral cargo. Gene expression While AAV...what will be delivered and expressed in the host cell from the transfer plasmid. Viral packaging and entry...altered tropism of AAV to target different tissue or cell types. AAV also requires some back up from adenovirus...promoter that you wish to deliver to your target cells. In adenovirus, the ITRs serve as a type of origin...deficient. This is typically supplied by the production cell line (HEK-293) during production. Fun fact: There...

.... PubMed PMID: 6276558. 2. Site-specific DNA recombination in mammalian cells by the Cre recombinase of...transgenic animals, embryonic stem cells, and/or tissue-specific cell types: Cre-lox recombination. What...downstream of promoters that are active only in certain cell or tissue types, during certain stages of development... recombinase can be expressed only in specified cells or at specified times. Combining this with some ...has been used to successfully introduce Cre into cells of interest. What if I need two separate recombination...

...3570-5. doi: 10.1073/pnas.1420294112. Epub 2015 Mar 2. PubMed. Find the plasmids from this publication ...toolkit in human cells. Nissim L, Perli SD, Fridkin A, Perez-Pinera P, Lu TK. Mol Cell. 2014 May 22;54(...advantageous to instead use a preexisting, conserved cellular machinery to process gRNAs. Hijacking tRNA processing...tRNAs are among the most abundant molecules in a cell, this system is clearly also robust enough to ensure...54(4):698-710. doi: 10.1016/j.molcel.2014.04.022. Epub 2014 May 15. PubMed.  Find the plasmids from this...

...JS, Legube G, Miller KM Genes Dev. 2015 Jan 15;29(2):197-211. DNA Cross-Bridging Shapes a Single Nucleus... R, Schmalhorst PS, Jude JG, Zuber J, Gerlich DW Cell. 2017 Aug 24;170(5):956-972.e23 Screen identifies... R, Schmalhorst PS, Jude JG, Zuber J, Gerlich DW Cell. 2017 Aug 24;170(5):956-972.e23 Rapid and phosphoinositol-dependent... Rando OJ, Xue Y, Swiderek K, Kuo A, Crabtree GR Cell. 1998 Nov 25. 95(5):625-36. An architectural role..., Ensminger AW, Fox AH, Chess A, Lawrence JB Mol Cell. 2009 Mar 27;33(6):717-26. Reporter-nanobody fusions...

...backbone, which expresses the gRNA from a Drosophila U6:2 promoter and Cas9 from the actin 5C promoter. Addgene...backbones for lentivirus LV 1-5 and retrovirus RV 2-5 were constructed. The authors then used GMAP assembly...engineering (MAGE) (1). MAGE uses recombineering (2) to simultaneously incorporate multiple single-strand...number of tandemly expressed gRNAs respectively) and 2) the size of the deletion. Smaller deletions generally... 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...

..., but at a lower efficiency (~20%). Figure 2: Schematic of multicomponent assembly with SLIC. Multiple...bacteria, so it can be transformed as-is into cells. The cells then get to work fixing up the “damage” and...nicked plasmid together long enough to get it into cells and let them do the repair work. The key is T4 DNA...

...-priced commercial devices.     Fig. 2: Workflow for open software (left) vs. open hardware (right...quantitatively evaluating antibody staining, or counting cells are now routine, open-source software projects that...portable device for studying plankton using flow cells, has become an established alternative to high-priced... and blueprints has the potential not only to accelerate scientific progress, but also to enable science...

...endothelial cell-pericyte, neuron-astrocyte, and cancer-stromal cell pairs.   Figure 2: Schematic...Winslow’s lab to label cells after cell-cell interactions. Using G-baToN, “sender” cells express GFP on the... and “receiver” cells express an anti-GFP nanobody on the cell surface. When the cells interact, GFP is...the receiver cell, and the receiver cell turns green. This system can be used in many cell types including...engineering with single-pot assembly Tracking cell-cell interactions New auxin inducible degron system....brgcm3sw     New system developed to track cell-cell interactions By Jennifer Tsang GFP-based Touching...of the interaction between the sender cell and the receiver cell. Image from Tang et al., 2020.   ...

...protocols 4.4 (2009): 495-505. PubMed PMID: 19300443. 2. Geraerts, Martine, et al. "Comparison of lentiviral...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...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...

...covered in more detail in part 2 of this blog series) controls the cell types in which the conditional...modified zygotes or embryonic stem cells. Modified embryonic stem cells are injected into a blastocyst which...engineered cells. Similar to transgenic mice, knockout mice are engineered during the embryonic stem cell stage...not all cells in the blastocyst were successfully edited by the injection of embryonic stem cells. Also,... interest into a zygote, or into embryonic stem cells that are then injected into a blastocyst. There ...of interest into a zygote or into embryonic stem cells. Non-homologous recombination incorporates this ...targeting vector is electroporated into embryonic stem cells and this will insert the gene at a specific site...

...perceived climate change risks. Nature Climate Change, 2(10), 732-735. doi:10.1038/nclimate1547 Kunda, Z. (...spend the afternoon lovingly tending to the stem cells I was using in an experiment, then spend the evening...

... solution can be stored at 4 °C for up to 2 months. After 2 months, discard the tube and thaw a new working... transfected using 1:1, 1:2, 1:3 and 1:6 µg of pRosetta :µg of PEI. The 1:2 and 1:3 ratios provided high...Plate 1x10 6 cells in a 75 cm 2 flask in a volume of 15 mL. Wednesday: Plate 1x10 6 cells in a T75 flask... transfect cells with a fluorescent plasmid using a variety of ratios. Check the cells 1–2 days after ...optimized for viral production) Day 1: Transfect Cells Day 2 (am): 18 h post transfection - Remove media, ...tissue culture plates. Incubate the cells at 37 °C, 5% CO 2 for ~20 h. Gently aspirate media, add 10 mL...Biological Safety Cabinet 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel...

...and open a new conversation about GMOs.  Challenge #2: Technical obstacles to plant synthetic biology In...challenging to engineer a specific function in these multicellular organisms. However, the rewards are great if...

... the transcriptional level.     Fig. 2 Tetracycline on/off promoters and modes of action ...force to control protein expression in mammalian cells: easy to use biological tools are abundant! In this...tag prior to introduction of the construct into cells. However, if you would like tunable control of an...

...processes that happen inside the cell,” says Pedre Pérez. Figure 2: Micrographs of the biosensor ...sensitive GFP2 (roGFP2)-based biosensors for live cells. His work focuses on the enzyme hydropersulfide-...biosensor expressed in the human cell line, Hep3B. From left to right: Mitotracker Red, GFP for the biosensor,...studies opioid use disorder. Her goal is to identify cell populations in the brain that contribute to the ...will explore the connectivity patterns of these cells and the functional contribution to a variety of ...administration and withdrawal to explore how these cells may encode the negative affect associated with drug...library of mutant sensors expressed in a mammalian cell line engineered to be electrically excitable. The...