...holes in the cell membranes of your sample to allow antibodies better access to intracellular targets. If...allowing antibodies into the cell where they could bind to intracellular epitopes. C) Permeabilizing with...method for visualizing molecules of interest within a cell or tissue. In that introduction, we broke down the... large proteins that need a little help crossing cell membranes as they are not able to diffuse across...target protein that is still on the inside of the cell. However, assuming you do need to permeabilize, ...For example, saponin interacts with cholesterol in cell membranes and leaves many membrane-associated proteins... organic solvents. However, detergents can lyse cells or cause you to lose soluble proteins from the sample...

...CRISPR-Cas System. Cell. 2015 Sep 23. pii: S0092-8674(15)01200-3. doi: 10.1016/j.cell.2015.09.038. PubMed...RNA-guided endonuclease of a class 2 CRISPR-Cas system." Cell (2015). PubMed PMID: 26422227.   Potential ...particularly helpful for targeting non-dividing cells, which are difficult to modify through homology-... Cpf1 orthologs known to be active in mammalian cells, LbCpf1 and AsCpf1. In both reports, on target editing... (RNPs) failed to induce off-target editing in a cell culture model. What we still don't know about Cpf1...reveals specificities of Cpf1 endonucleases in human cells. Nat Biotechnol. 2016 Jun 6. PubMed PMID: 27272384...specificities of CRISPR-Cas Cpf1 nucleases in human cells." Nature Biotechnology (2016). PubMed PMID: 27347757...

...metabolite in a cell. The only way to do that at scale right now is to grow up a bunch of cells, sacrifice ...capable of storing data and computing inside living cells. He's also a co-founder of the BioBricks Foundation...as an operating system for engineering an entire cell. Typically when people use plasmids, they are trying... you could imagine engineering the entirety of a cell. That's a big long-term technical challenge. We ...That's central to everything that goes on inside a cell. Until we get a handle on precise, forward engineering...scale, it's going to be tricky to tame the rest of a cell. The BIOFAB plasmids represent the first systematic...transistor-like digital genetic switches that enable cellular computing. These logic gates built from transcriptors...

...Collection The Armamentarium for Precision Brain Cell Access is developing a collection of molecular genetic... reagents to gain access to many different brain cell types. The expanded collection of Armamentarium ...mCherry-tagged channelrhodopsin-2 driven by PV+ basket cell-targeting enhancer Gordon Fishell Viviana Gradinaru...construct expressing dTomato driven by PV+ basket cell-targeting enhancer Gordon Fishell Viviana Gradinaru...mCherry-tagged channelrhodopsin-2 driven by chandelier cell-targeting enhancer Gordon Fishell Viviana Gradinaru...construct expressing dTomato driven by chandelier cell-targeting enhancer Gordon Fishell Viviana Gradinaru...

...gives a talk at the Cell Symposia: Gene- and Cell-based Therapies: CRISPR, Stem Cells, and Beyond meeting...III product is produced and can infect bacterial cells. If a phage does not deliver an active variant, ...passage from the infected to a fresh uninfected host cell culture). This dilution step represents the major...but gets manually passaged from an infected host-cell culture to an uninfected culture which results in...

...to quantify rates of mutation in a bulk or single cell-derived genomic DNA sample as part of a precision... successfully analyzed gDNA from a few thousand cells from single wells of a 96-well plate without prior... that they are easily able to distinguish single-cell derived samples with mono-allelic mutations versus...Isolation of single-base genome-edited human iPS cells without antibiotic selection. Nat Meth 11, 291–293... and Highly Specific Screening of Genome Edited Cells. PLoS ONE 11, e0153901–17 (2016). PubMed PMID: 27089539...mutagenesis induced by CRISPR-Cas nucleases in human cells. Nature Biotechnology 31, 822–826 (2013). PubMed...

...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...

...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...2. Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Sauer...

...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...optical live imaging of proteins, organelles, and cells. Similar to FPs, PA-FPs allow for non-invasive labelling...dynamics of mitochondria in healthy and apoptotic cells (9). PA-FPs have helped to revolutionize the field...

...mammalian cells. Harrington et al tested GeoCas9’s editing skills by transfecting HEK293 cells with GeoCas9...choice for a number of model organisms: mammalian cells, yeast, drosophila, plants, worms, zebrafish, frogs... 45 °C 50 - 70 °C Genome Editing in Mammalian Cells? Yes Yes Longevity in Serum?* No Yes Table ...experimental results. GeoCas9 RNP editing in mammalian cells Besides being able to take some heat, GeoCas9 can...

..., or express the insert they carry in the target cell. These vectors allow you to test the function of...If you are planning to track your protein in the cell or purify it – and you do not have a specific antibody...selection cassette to screen for positive transfected cells. The addition of an in-frame fluorescent fusion ...get away with generating transiently transfected cells, you will not need a mammalian selection cassette.... Other vectors can be expressed in fly culture cells like S2 and carry the metallothionein promoter (...

...allows you to sort for GFP-positive cells and to enrich for those cells that were positively transfected....introduction of a mutation into the genome, how can cells with that mutation be selected/screened? A4: Before...can use a selection marker to select transfected cells (for example, plasmid with a puromycin resistance...paper). If you detect positive HR, isolate single-cell colonies, grow them up, then perform individual ...

...important to normal cell signaling and physiology, and, when dysregulated, can enable cancer cell survival. Although...that is essential for regulating the growth of the cell. p53 p53, the most frequently mutated gene in human...

...temperature incompatible to genome editing in human cells. The Feng Zhang lab identified a Cas12b from Bacillus...can block Cas12a-mediated genome editing in human cells. Read the paper in Science Find the anti-CRISPR...anti-CRISPR expression plasmids CRISPR-based method for cell lineage tracing Valcyrie is a CRISPR-based marker...marker excision method for marking distinct cell clones developed by the Michalis Averof lab. It’s an alternative...

...construct was then introduced to HeLa cells (Figure 1). The resulting HeLa cells show robust labeling, with strong...protein localization, as well as cellular processes like gene expression and cell migration (Sladitschek and... MXS-chaining were used to label subcellular structures in live cells. Four individual expression constructs...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...applications in flow cytometry approaches in mammalian cell culture systems (their original goal). Their work...detection of each fluorophore at the respective subcellular structure (Figure 1). Here, MXS Chaining gave...

... primary cells. The authors used this platform to assay dozens of GPCRs across many cell types in...biosensor platform. Cell, 187(6), 1527–1546.e25. doi: https://doi.org/10.1016/j.cell.2024.01.028. New...Prime editing (PE), initially developed in human cells, has been uniquely challenging to apply in dicotyledonous...labeling approach using these constructs in HEK 293 cells (Smith et al., 2024). They then used single-molecule..., CLIPf-, and CLAP-tagged proteins for mammalian cell expression. These plasmids will be useful not only...recordings of spontaneous activity in human stem cell-derived neurons expressing ASAP5-Kv. C) Cultured...

...(FRET) microscopy imaging of live cell protein localizations. J Cell Biol. 2003 Mar 3;160(5):629-33. ...measurement of single molecule interactions in living cells using a microscope. Historically, molecular interactions...suitable for studying interactions within living cells. By coupling fluorophores to proteins, pioneers ...directly detect protein/protein interactions in living cells. Since then FRET has also been used to measure conformational...efficiency and cross-talk. Methods to Measure FRET for Cell Biology Studies Several methods have been used over...microscopy: monitoring protein interactions in living cells. Bioessays. 2012 May; 34(5):341-50. doi: 10.1002... Constructs to target mTurquoise2 to various subcellular compartments pCEP4CyPet-MAMM Cyan Mammalian...

...to activate programmed cell death or dormancy for phage-infected bacterial cells so as to limit the spread...sequence. DSBs are preferentially repaired in the cell by non-homologous end joining (NHEJ), a mechanism...PAM site. When a repair template is present, the cell may repair a DSB using homology-directed repair ...Cas protein and a target-specific gRNA in the same cell at the same time. Single plasmids containing both...

...to activate programmed cell death or dormancy for phage-infected bacterial cells so as to limit the spread...population. Type VI enzymes that function in mammalian cells can be used to attentuate RNA levels. In mammalian...

...producer cell line, such as HEK-293T, production of viral particles, and transduction of a target cell line...re derived from viruses that can infect people, cells, and animals. Some of these viruses are completely...While the virus can still enter (and thus infect) cells, this safety feature prevents the virus from replicating...replicating and spreading in non-target cells, including yourself as the researcher.   Figure 1:... the procedure. Mice move much more quickly than cell cultures do, and tired researchers are accident-...vector carries an shRNA to a gene essential for cellular function, this would also be a higher risk vector...