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...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 (...

...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...

...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...

...proteins that bind an RNA motif of interest in living cells. Challenges with existing RNA-protein interaction...and naturally occurring complexes present in the cell, it can also introduce non-physiological RNA-protein...a short timescale, such as during drug action or cell signaling. Limitations of RaPID Like other BirA*... controls include both scrambled RNA motifs and cells transfected with BirA* protein alone (i.e. no RNA...protein that’s highly expressed in neural progenitor cells. Knock-down of QKI decreased Zika viral RNA levels...2018). RNA–protein interaction detection in living cells. Nature Methods, 15, 207-212. PubMed PMID: 29400715...

...chemically. Enzymatic biotinylation occurs naturally in cells and is the process by which biotin is covalently... Immunosorbent Assay) Southern/Northern blotting Cell surface labelling Attaching polypeptides to a substrate...substrate Flow cytometry/fluorescence-activated cell sorting (FACS) In situ hybridization Methods for...proteins in bacteria, yeast, insect and mammalian cells. Check out Kay et al., 2009 for methods on biotinylating...Application of avidin-biotin technology to improve cell adhesion on nanofibrous matrices.” Journal of Nanobiotechnology...purification of tagged transcription factors in mammalian cells and transgenic mice.” PNAS 100.13 (2003):7480-5....-H., Lee, C.-Y., & Li, P.-C. “Buoyancy-Activated Cell Sorting Using Targeted Biotinylated Albumin Microbubbles...

... of human stem cells when students are not even allowed to manipulate prokaryotic cells? Especially, if...where you need to apply basic molecular biology, cell biology, microbiology, genetic engineering and strategic...could be used to genetically manipulate human stem cells. Then they had 10 minutes to expose their strategy...construct and explain why they selected a certain target cell population. Who is the winner? Ultimately, society...

... of all of the mRNA bound-ribosomes in a cell. When cellular mRNAs are digested with ribonucleases, ribosomes...the effects of 5’ UTR variants in translation of cell cycle protein Emi1, showing that ~80% of transcripts...powerful tools for studying translation in various cell types and physiological states. References 1. Ingolia...imaging the first round of translation from single cells to living animals.” Science 347(6228) (2015): 1367...of Translation of Single mRNA Molecules In Vivo.” Cell 165(4) (2016): 976-89. PMID: 27153498 PMCID: PMC4889334...Regulating translation is key to cellular function, especially during development or stress. With ribosome...explore the complexity of translational control in cellular physiology. Ribosome profiling Ribosome profiling...

... between Cas9 (which causes the breaks) and host cell DNA repair factors (which repair those breaks) makes...example, correcting the allele that causes sickle cell anemia. However, HDR occurs much less frequently...a structure could drive sequence replacement in cells. Targeting three dCas9 molecules to a precisely ...functional Cas9 protein and the targeting guide RNA into cells by electroporation. Results from our lab and others...Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins...

... yeast, plant, insect, and mammalian cells, including human cells. piggyBac is biased to integrate at ...is active in vertebrates and integrates in human cells at rates similar to retroviral vectors. The hyperactive...piggyBac has an integration efficiency in human cells similar to retroviral vectors. The hyperactive PB...transposase (hyPB) is ~10-fold more active in mammalian cells compared to the codon-optimized wild-type piggyBac...G/C)TNA. Tol2 can deliver 10-11 kB to mammalian cells without decreased efficiency, with a maximum cargo...and has lower efficiency of integration in human cells than piggyBac and Sleeping Beauty. Minimal Tol2 ... or barcodes. When transposons are delivered to cells or a model organism, they insert into the host’s...

...can be used in bacterial and cell-free expression systems, mammalian cells, yeast, flies, and even plants...transport studies in HEK cells, recombinant protein purification in bacterial cells, and a two-hybrid protein...for synthetic biology applications in bacteria or cell-free systems. The approach is particularly useful...add-on kit and is compatible with E. coli and/or cell-free systems. Protein expression in yeast MoClo ...of expression and integration needed in mammalian cell types, and comes with a large library of common ...protein interaction study in yeast cells. Need more MoClo? Check out Addgene's MoClo Guide! Customizing...Construction of Biological Circuits in Mammalian Cells.” ACS Synthetic Biology, vol. 8, no. 11, American...

...stain the cells that contain this mRNA-riboprobe-antibody complex and, as a result, these cells turn blue...specific cell types or entire organs in planarians. For example, the gene Piwi labels neoblast cells and the...techniques to alter gene expression or to label specific cells, but there are too few resources to teach students... in tissue. This is useful for labeling specific cell types and for viewing how gene expression changes...

... be introduced into host cells, such as bacterial, yeast, or mammalian cells, for antibody production ...Nanobodies and recombinant binders in cell biology. Journal of Cell Biology 209:633–644 . https://doi.org...animals and bleed dates and even clonal hybridoma cell lines can produce more than one mAb (Bradbury et...plasmid or from an integrated sequence in a stable cell line. The genes encode the heavy chain and light...antibody protein binds an antigen. Image from Fvasconcellos.     How are Recombinant Antibodies Made...

...circuits to be deployed out of the laboratory in a cell-free and biosafe format that was stable for up to...possible and submitted our manuscript to the journal Cell just six weeks later. The Zika publication timeline...of Toronto. His research is focused on developing cell-free synthetic biology applications for human health...Virus Using Programmable Biomolecular Components." Cell (2016). PubMed PMID: 27160350. Find Plasmids from...de-novo-designed regulators of gene expression." Cell 159.4 (2014): 925-939. PubMed PMID: 25417166. PubMed...Keith, et al. "Paper-based synthetic gene networks." Cell 159.4 (2014): 940-954. PubMed PMID: 25417167. PubMed...

...Generating a no-fuss genetic knockout  Knock-out cells or animals are engineered by introducing Cas9-mediated...transcription by blocking its initiation. In mammalian cells, robust repression requires targeting dCas9 along...study essential genes whose knockout would cause cell lethality. The Krüppel associated box (KRAB) is ...repressing target genes in a variety of species and cell types can be found on Addgene’s website. Activating...activating target genes in a variety of species and cell types can be found on Addgene’s website. ...optimized for expression in different species and cell types including (but not limited to) bacteria, yeast... have delivered Cas9 and a sgRNA to your target cells, it is time to confirm that your target sequence...