... genetic loci within a single cell. One might imagine transfecting cells with both the nuclease-positive...while its nucleolytic activity is eliminated (Fig. 1) (1, 3). The gRNA can be further modified by incorporation...nuclease-competent Cas9, obviating the need to generate new cell lines or transgenic animals expressing dCas9 as ...reliance on orthogonal Cas9 species. Within a single cell, full-length guides targeting one set of genetic...knockin mice for genome editing and cancer modeling." Cell 159.2 (2014): 440-455. PubMed PMID: 25263330. PubMed... Podcast Interview with Alex Chavez Figure 1: Schematic of native (nuclease-competent) Cas9 interacting...which Cas9 protein is directed to which target site (1). Although this is possible, this strategy suffers...

...live-cell imaging, and mechanobiology: seeing is believing." Annu. Rev. Biomed. Eng. 10 (2008): 1-38. ...discovered such as Kaede and PS-CFP2 (see Table 1). Table 1: Properties of Selected Irreversible Photoactivatable...continuously synthesized and degraded within the cell, so at any given time, an FP-chimeric protein may...Since emission of green light is more phototoxic to cells, most scientists working on in vivo imaging models... this is not ideal for experiments in mammalian cells. Dendra2 is another monomeric PA-FP which unlike...tetrameric. Tetrameric PA-FPS are better for whole cell imaging and studying organelle trafficking. Monomeric...the photoactivation step can actually harm your cells before your experiment even begins! If you are performing...

...mitophagy in mammalian cells using a single fluorescent biosensor. Scientific Reports,5(1). doi:10.1038/srep12397... Mammalian Autophagy Research. Cell,140(3), 313-326. doi:10.1016/j.cell.2010.01.028. PubMed PMID: 20144757...analyzing autophagy of cytosol and organelles in yeast cells. Autophagy (Greek for “self-eating”) is induced ...radio-labeled long-lived proteins as a proxy for autophagy. Cells are cultured with isotope-labeled amino acids for...fluorescence regardless of its localization in the cell. SEP’s pH sensitivity means Rosella will fluoresce...’s fused to the C-terminus of DsRed.T3. See Table 1 for a summary of Rosella’s excitation and emission...fluorescence Rosella emits based on its localization. Table 1: Summary of Rosella’s excitation and emission spectra...

... defined population of cells (e.g., a neuron subtype) and specific subcellular compartments for measurements...calcium concentrations and transients in living cells and organisms. Unlike chemical indicators, these...simultaneous (1) user-controlled illumination with violet light and (2) exposure to increased intracellular calcium...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 ...measure changes in calcium specific to the neuronal cell body or the axon (GCaMP6 only). The genetic elements...long-term expression and integration within the target cells, ensuring more consistent and reproducible results...

...delivered using AAV injection, the majority of the cells surrounding the injection site will become infected... ensure that the reporter remains silent until a cell or tissue-specific Cre is provided. Since the FLEx...used to study the physiology or behavior of certain cell populations.  Acknowledgement:Through a partnership...FLEX switch targets Channelrhodopsin-2 to multiple cell types for imaging and long-range circuit mapping...coding sequence in the antisense orientation (Figure 1, top). The entire DNA cassette would be flanked by...include both Cre and FLP recombinases.   Figure 1 (modified from Schnutgen et al. Nat Biotech 2003):...identical sites on one end of the DNA fragment (Figure 1, middle). A second recombination event then excises...

...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...

...epitopes likely to be presented by T cells to elicit an immune response. T cell epitopes are found in either ...that take place within an antigen-presenting cell like a T cell. It is also not yet possible for these algorithms...traditional 2D assays with immune cell lines or peripheral blood mononuclear cells (PBMC) in 3D models of the...CD4+ or CD8+ T-cells or monocytes. PBMCs can be further differentiated into dentritic cells or macrophages...impurities in the formulation. Efforts to predict T cell epitopes focus on characterizing those epitopes ..., that are commonly used for the prediction of T cell epitopes based on the amino acid sequence of the...lymphatic system. PBMCs consist of various immune cells, and can be used as a whole or can be used to isolate...

...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...overcomes many of those limitations (Figure 1). Figure 1: Opto-katanin induces fast, localized microtubule...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...

...Fluorescence Activated Cell Sorting (FACS), allowing for enrichment of successfully targeted cells that can be used...opportunities for investigating genetic mechanisms related to morphogenesis, cell fate specification, and human ...organoid model (Sun, et al. 2021).  Figure 1: Example schematic from the Organoid EasyTag system...without altering that of the internal control (Fig 1). Because this method relies on the fluorescence ratio... a major economic impact.   Design and mechanism of action of the receptor-based SL fluorescent...

...luciferase assays require cell lysis as the most efficient means to disrupt the cell membrane and deliver ...miRNAs for effectiveness. Analyze Cell Signaling Pathways Known cellular signaling pathways can be investigated...luciferase as a genetic reporter in plant and mammalian cells. Luciferase assays have since become a gold standard... serves as an indicator of transcription within cells, where detection of the reporter protein or its ... into the desired mammalian, plant or bacterial cells. Expression of luciferase as the reporter gene is...can permit measurements of luminescence from live cells. Monitor miRNA knockdown Expression of luciferase...codon, which is then transfected into the desired cells. A hybrid mRNA transcript containing luciferase ...

...Deletions in Human Embryonic Stem Cells Using Type I CRISPR-Cas. Molecular Cell, 74(5): 936-950.e5 (2019). 10.1016...between Cas7-11 and Cas13 are 1). non-specific RNA cleavage and 2). cell toxicity. Cas7-11 does not display...a class 2 CRISPR-Cas system. Cell, 163(3): 759-771 (2015). 10.1016/j.cell.2015.09.038 Harrington, B., ...cleavage and is significantly less toxic to mammalian cells than Cas13 (likely due to the lack of collateral... targeting single stranded RNA.   Table 1: Fast facts on Cas family proteins and genome editing... which target and cleave RNA through the same mechanism as traditional spCas9. If you are looking to modify...it perfect for targeting mRNA and useful as a mechanism of transient knockdown of gene expression. This...

...With regard to target cells or organism, some primary cells and differentiated cell types are picky when...packaging plasmids into a viral production cell line, usually HEK-293 cells. When all of these viral plasmids ... rogue HEK-293 cell). This virus-containing media can then be placed on whatever cells you would like ...just zapping your cells? That really depends on two things. First, what type of cell or organism context... depending on the virus, cell type, and/or desired event.   Figure 1: Transduction to expression...methods involve DNA incorporation into the cell, but the mechanisms vary. Why transduce? So why use a virus...Transductions utilize the ‘normal’ mechanism of viral infiltration into a cell. The twist is that the typical...

...human T cells and hematopoietic stem and progenitor cells (HSPC)s to ~40% expression in the K562 cell line...gene to airway epithelial cells in mice. This vector transduced ~10% of cells, similar to the transduction... then independently packaged into an AAV. When a cell is co-infected with both part A and part B AAVs,... always.  The combination of serotype and tissue/cell type could have a big impact on the rate of transgene...since both of these variables influence whether cells will be co-transduced with multiple copies of both... followed by the 3’-end of the transgene. When a cell is transduced by both viruses, if the AAV genomes...optimal sequences, the group achieved ~80% of muscle cells expressing mini-dystrophin.  3. Hybrid  Overview...

...also acceptable. Aims should be mechanistic, doable by 1-3 people in 1-5 years, and contribute to resolving...example, "while it is possible to grow liver stem cells and liver-like mini-organs or organoids, neither...combine bioengineered scaffolds with patient stem cells to generate compatible functional livers.” Why are... optimization of fluorescent proteins,  and live cell imaging approaches. Erik is also an avid long distance...possible, do at least two full length practices (~1 h) and get feedback. It’s also helpful to practice...an impediment to progress in a field. It is a mechanistic problem, a "how" question, not a vague goal or...ambitious goals and can help you distinguish mechanistic questions from open ended screens and data collection...

...to the most colonies after transformation. When a 1:1 vector:insert ratio is used in the DpnI digestion...properly. Directly transform chemically competent cells with the digested mixture. There is no need for ...purification before transformation. After transforming cells, you should perform colony PCR with vector specific... get tips for applying it in your own lab. Table 1: Comparison Between Standard Restriction Cloning and...Connecticut interested in protein science.     References: 1. Celie, Patrick HN, Annabel HA Parret, and Anastassis...PCR cloning method." BMC biotechnology 11.1 (2011): 1. PubMed PMID: 21992524. PubMed Central PMCID: PMC3207894...appropriate antibiotic. Although the precise mechanistic details of the process are not well understood...

...transformation efficiency of the strain: when asking the cell to perform multiple recombination events, all linear...linear fragments need to get into the same cell. For simple cloning requiring just one or two recombination...recombination events, homemade competent cells of 107 CFU/μg are perfectly fine, yet performing multiple modifications...modifications will require ultracompetent (109 CFU/μg) cells. Primer design Using specific primer design (Figure...two-step, 2 hr protocol prior to transformation (Figure 1). As this approach requires no special reagents or...with linearized vector backbone. DpnI digestion - 1 μl DpnI is added to the PCR mix to remove parental...confirm primer design.  Use minimal template DNA Use 1 ng of PCR template DNA to minimize false positive ...

...dual-color control of neurons. Nature Communications, 12(1), 1–20. https://doi.org/10.1038/s41467-021-24759-5 Zhang...pulses to the subject/cells expressing the optogenetic tool causing a change in cellular activity or gene ...involving many downstream signaling mechanisms that are often cell-type specific. If it is something you...positively charged ions to rapidly pass through the cell membrane and initiate an action potential - a very...such as Na+, K+, and Ca++ to passively enter the cell with the concentration gradient to depolarize it...the concentration gradient and hyperpolarize the cell (Mattis et al., 2012). Bicistronic vectors combine...different wavelength, turns it off, allowing the cell to be excited over a sustained period of time (Gong...

... edit A → Y (Y = T or C) in 67.17% of screening cells (Tong, Wang, et al., 2023). Based on this promising...Cas9 nicks the non-edited strand to encourage the cell to repair the DNA using the edited strand as a template... efficient A•T-to-C•G base editing in mammalian cells and embryos. Nature Biotechnology, 42(4), 638–650...programmable T-to-G and T-to-C base editing. Molecular Cell, 84(7), 1257-1270.e6. https://doi.org/10.1016/j....efficient T-to-G and C-to-G editing in mammalian cells. Nature Biotechnology, 42(10), 1538–1547. https:...a glossary at the end of this post! Figure 1: Base transitions are edits between the purine bases...uracil DNA glycosylase. Nature Communications, 15(1), 4897. https://doi.org/10.1038/s41467-024-49343-5...

...building it! Table 1 has a list of Fragmid destination vectors currently available. Table 1: Fragmid destination...Drosophilia Plasmid pRDB_051 (for stable fly cell lines; attL and attR sequences for genome integration...Drosophilia Plasmid pRDB_052 (for stable fly cell lines; Hygromycin resistance gene; for genome integration...rapid assessment of emerging CRISPR technologies. Cell Genomics, 4(3), 100519. https://doi.org/10.1016/...components on the Fragmid website.   Figure 1: (Top) Schematic overview and timeline of the Golden... CRISPR vector the Fragmid way!    Figure 1: Selecting your vector type on the Fragmid website...components available will depend on your answers in step 1, so you’ll only be able to select components that ...

...at lower copy numbers within bacterial cells, and bacterial cells with multimers grow more slowly compared...stable and more prone to being lost in bacterial cells compared to monomers, although this can be prevented...increase the frequency of base substitution in human cells. J Biol Chem. 2011 Mar 25;286(12):10017-26. doi:...plasmids leading to the formation of plasmid multimers. Cell. 1976 Dec;9(4 PT 2):707-16. doi: 10.1016/0092-8674...supercoiled plasmid DNA concatemers in mammalian cells. J Gene Med. 2009 May;11(5):444-53. doi: 10.1002...essential for plasmid monomerization and stability. Cell. 1984 Apr;36(4):1097-103. doi: 10.1016/0092-8674...indicates the presence of a multimer.   Fig. 1: The monomer of Plasmid 12259: pMD2.G (depicted on...