... ml per site) Skill required: beginner Intradermal Intradermal (ID) injections occur between the layers...”, or injections that are easy to pick up with minimal practice after initial mouse training. Other techniques...like the back) is preferred. A common use of intradermal injections is studying inflammatory responses...method is useful for analyzing localized events. Intradermal injections require a bit of extra prep, as the...disruptive, but surprisingly, it is considered minimally invasive. Restraint: anesthesia Location: spinal...intubation of the mice, which can be tricky given their small size. This technique is most often used for viral...muscle 25–27 < 2–3 mL (~1 mL per site) Beginner Intradermal Anesthesia Between skin layers 26 < 0.05 mL Advanced...

...replicate the plasmid in addition to its own chromosomal DNA, costing additional resources to maintain...selection in non-bacterial cells such as yeast or mammalian cells.   Name Class Mode of Action*   Working...Concentration** Kanamycin aminoglycoside Binds 30S ribosomal subunit; causes mis-translation Bactericidal 50.../mL Spectinomycin aminoglycoside Binds 30S ribosomal subunit; interrupts protein synthesis Bactericidal...100 ug/mL Erythromycin macrolide Blocks 50S ribosomal subunit; inhibits aminoacyl translocation Bacteriostatic...100 ug/mL Tetracycline tetracyclin Binds 30S ribosomal subunit; inhibits protein synthesis (elongation...Bacteriostatic 10 ug/mL Chloramphenicol   Binds 50S ribosomal subunit; inhibits peptidyl translocation Bacteriostatic...

...greyscale.   ARG expression in mammalian cells The first-generation mammalian ARGs (mARGs) enabled ultrasound... focused on bacterial ARGs and Tom focused on mammalian ARGs. ARG expression in bacteria The first-generation...ultrasound visualization of in situ mammalian gene expression in deep tissue. However, not unlike the first-generation...second-generation mARGs with higher expression, a smaller genetic footprint, and nonlinear acoustic scattering...flos-aquae gas vesicle gene cluster because of its small size and its ability to produce gas vesicles with...expression. We were able to express Anabaena GVs in mammalian cells without the shell stiffening protein GvpC...various cell lines from different tissues and mammalian species to produce gas vesicles. New mARG plasmids...

...antibodies are derived from animals, such as rabbits or goats; the animals are injected with the antigen...of the target protein: instead, it recognizes a small portion of the protein, known as an epitope. Epitopes...recognize. This triggers an immune response within the animal, and its B cells will churn out antibodies against...injected protein. Several weeks later, blood from the animal is drawn and the antibodies are extracted. These...multiple epitopes on the protein. However, each animal will make antibodies against different protein ...by fusing B cells that have been extracted from animals injected with protein antigen to myeloma cells.... single epitope of the target protein. However, small mutations in the DNA of the hybridoma can occur,...

...Transgenic animals can be made by simply micro-injecting DNA into the worm, such as animals that express...musculus) Let’s start with the most preferred mammalian model organism of many researchers: the mouse ...Mus musculus). Mice have many advantages as a mammalian model organism for scientists as they have a relatively... relatively short generation time for mammals - the time between being born and giving birth - of about... a set of modular vectors that can be used to optimally control gene expression in specific cells of the...in zebrafish (Howe et al., 2013). Zebrafish are small, easily maintained as they are housed in large groups... to edit their genomes or to create transgenic animals. Zebrafish are an ideal model organisms for embryonic...

...and ALK) by linking small molecules that bind these target proteins to other small molecules that bind...polyubiquitination of the target protein and proteasomal degradation. Remarkably, small molecule degraders provide distinct...than inhibiting protein function, these so-called “small molecule degraders” recruit the proteasome to destroy...destroy targeted proteins. We previously developed small molecule degraders that achieve selective degradation...this technique to oncoproteins that lack selective small molecule binders. To overcome this limitation, we...dTAG system The dTAG system pairs highly selective small molecule FKBP12F36V degraders (dTAG molecule) and...Chemical Biology for examples). Monitor neo-substrates Small molecule degraders may recruit CRBN to nonstandard...

... gene encodes a vacuolar alkaline phosphatase. Normally PHO8 is synthesized at the ER, delivered to the...Atg8p (“p” stands for protein) associates with autophagosomal membranes, so tagging it with GFP allows for...tracking the localization or accumulation of pre-autophagosomal structures, autophagosomes and autophagic bodies...bodies in yeast. A GFP-tagged version of LC3, the mammalian Atg8p homolog, can also be used to monitor autophagy...4.9 - 9 SEP 488* nm 508 nm ~6.5 - 9 *Optimal sequential excitations wavelengths as determined...synthase’s mitochondrial targeting sequence. Under normal growth conditions, both variants are excluded from...measurements of macroautophagy and mitophagy in mammalian cells using a single fluorescent biosensor. Scientific...

...expression. Transient episomal delivery Non-integrating methods have a smaller genetic footprint compared...are flanked by loxP sites. While there is only a small loxP scar behind at the site of excision, this approach...cut and paste” mechanism between a vector and chromosomal DNA. PB has a cloning capacity of ~9-14 kb. This...protein delivery considered the safest since there’s minimal risk of lingering expression of reprogramming factors...expressed and they don’t integrate or replicate. Their small size leads to higher transfection efficiencies and...DNA-silencing mechanisms. While minicircles are typically smaller than traditional plasmids, Jia et al generated ... and allows for replication of the plasmid in mammalian cells and assists in tethering the vector to the...

...Polyclonal antibodies produced in animals may be variable among animals and bleed dates and even clonal ...1: (Top) Polyclonal antibodies are produced in animals and consist of a mixture of antibodies recognizing...used just as you would use antibodies made from animals or hybridomas.   Figure 2: (Left) Figure ... display, yeast display, ribosome display, or mammalian display (Tsuruta et al., 2017). In vitro selection...antibodies to targets that may not work well in animals because of similarities to host proteins. After...into host cells, such as bacterial, yeast, or mammalian cells, for antibody production and subsequent ...genetically drift over time. Antibodies made from animals are limited in supply and producing antibodies ...

...express siRNAs of their choice in mammalian cells.  Learn more about Mammalian shRNA Tools for RNAi! Cre-Lox...control of Murine Stem Cell retrovirus promoter for mammalian gene knockdown. Vector provided by the Qi lab....can be used for in vivo phenotypic screening of small-molecule libraries and can be combined with high-throughput...library to support functional investigations of the mammalian genome that can reveal how genetic alterations...introduction of multiple genetic alterations into mammalian cells. The MuLE system kit from the Frew lab consists... or enzymatic reporters for cellular assays and animal imaging studies. The ability to simultaneously ...high throughput genetic modulation studies in mammalian cells and tissues. This modular and flexible system...

...implement, they can be highly specific with a small and minimally-disruptive genetic tag on the protein of ...show good specificity for their own ligands with minimal cross-reactivity. Other strategies include using...Benzylcytosine (BC) Some Easy Ligases Small, varies Various Few Complicated Non-natural...CLIP-tag (both 182 aa), and eDHFR (159 aa) are smaller than HaloTag (295 aa) – for comparison, common ...fluorescent proteins are typically 230–250 aa. Smaller size isn’t a guarantee that a tag won’t disrupt... fusion protein is functional. In some cases, a small peptide tag like tetracysteine might be the only...for the covalent labeling of fusion proteins with small molecules in vivo. Nature Biotechnology, 21(1), ...

...templates in somatic cells of adult animals in order to create animal models for particular cancer. That...isogenic AdVs encoding TALENs targeting the same chromosomal region. The CRISPR/Cas9-derived RNA-guided nuclease-induced... will interact with sporadic double-stranded chromosomal DNA breaks “that always happen naturally.” These...Gonçalves says that advantages of AdVs include their episomal nature and very efficient introduction of DNA ...into therapeutically relevant, non-transformed mammalian cells. These viral vector systems also work equally... well in dividing and quiescent, post-mitotic mammalian cells. Applications of CRISPR delivery by adenoviral...be useful to labs wishing to create this kind of animal models. Start using adenoviral vectors with your...

...problem is to tag the protein of interest with a small biochemical “epitope tag” such that these tagged... insertion of epitope tags at target genes in mammalian cells (Bressan et al. 2017; Liang et al. 2017)...Cas9 protein-based method for epitope tagging in mammalian stem cells. For easy scalability to the 96-well...Ribonucleoprotein (csRNP) delivery Epitope tagging of mammalian genes using CRISPR depends on successful delivery... 96-well format and our csRNP method suits both small and medium-throughput epitope-tagging experiments...experiments, it can be ordered in bulk and stored in small aliquots at -80 oC. Assemble csRNP: Anneal crRNA... function of large protein cohorts in diverse mammalian cell types. A detailed step by step protocol for...

...approximation of reward or the sense of pleasure that the animal perceives through taking a drug. The ideal behaviors...reinforcement (instills a sense of “reward”), then the animal will repeatedly nose-poke at the active hole [1...distinguished visually through different patterns and the animal only receives stimulation in one of the two chambers... image below). If a circuit is reinforcing, the animal will prefer to spend most of its time in the chamber...behavioral experiments. Comparison of c-fos amongst animals that receive different types of stimulation may...any type of behavior analysis, simply take a few animals and stimulate them for 30min to an hour and observe...stimulation of a particular brain region may affect the animal; stimulation could induce seizures or some other...

...interacting partners and perform whole animal screens for small molecules that block the neurodegenerative...future small molecule screens. He plans to work with a partner at IRCCS Besta in Milan to test small molecules...fail to garner much international attention. Their small numbers do not, however, make these patients suffer...these studies will accelerate the development of small molecules for use in the clinic and thereby restore...physicians and scientists.  Although the form of malignant mesothelioma that affects the lining of the lungs... they will use to develop diagnostic tools and animals models of the disease in partnership with Taconic...

...of the most common tags available. Maltose binding protein Maltose binding protein (MBP) is a 45 kDa tag...naturally occurring histidine-rich proteins in mammals. Mammalian purification is still possible but requires...within the expression system (bacteria, yeast, mammalian, etc.). To individually isolate a protein, there...more straightforward and of a higher purity than mammalian purifications due to the presence of more naturally...with several different resin types and is a fairly small tag that is unlikely to perturb protein function.../0471140864 Fox, J. D., & Waugh, D. S. (2002). Maltose-Binding Protein as a Solubility Enhancer. In P....

...spatial scales from sub-cellular to whole-body in small animals, such as mice (Matlashov et al., 2020). Enhanced...intensities in NIR. Usually, mercury lamps are suboptimal, metal halide lamps are better, and xenon lamps...combined in two-color and multicolor NIR imaging.  Small miRFPnano A separate class of NIR FPs, named miRFPnanos...engineered to incorporate BV. Their advantages are their small size (17 kDa for miRFPnanos, 35 kDa for miRFPs, ...aExtinction coefficient. bQuantum yield. cDetermined in mammalian cells. dDetermined as effective NIR fluorescence...cells with no supply of exogenous BV and after normalization to fluorescence of co-transfected EGFP. e Originally...Brightness of Near-Infrared Fluorescent Proteins in Mammalian Cells. Cell Chem Biol 24, 758-766 e753 (2017)....

... by plants, most bacteria and some fungi, thus mammals must obtain the vitamin through external sources...Humans absorb most of the biotin they need in the small intestine from the microflora that reside there.....   In the late 1930s it was observed that animals fed a diet of primarily egg whites would develop...B7/biotin. Egg white injury would plague these animals even if their diet was being supplemented with ...biotin in molecular biology Biotin is a relatively small, water soluble molecule that does not interfere ...expressing proteins in bacteria, yeast, insect and mammalian cells. Check out Kay et al., 2009 for methods ...Hu, J., & Zhu, Y. “A non-radioactive method for small RNA detection by northern blotting.” Rice 7.1 (2014...

...ligase fusion protein identifies proximal and interacting proteins in mammalian cells." J Cell Biol 196.6 (...proteins per cubic micron in bacteria, yeast, and mammalian cells (Milo, 2013). The number of interactions...also currently available at Addgene.  BioID2: A smaller improved promiscuous biotin ligase The Roux Lab...Lab in 2016 optimized BioID to create BioID2, a smaller and improved promiscuous biotin ligase (Kim et ...biotinylation. Compared to BioID, BioID2 exhibits enhanced proximal labelling, requires less biotin, and allows scientists...hypothesized that the low sensitivity was due to suboptimal folding or stability. Thus they conducted multiple... a monoglycine residue at the N terminus with a small peptide (LPETG). In EXCELL, the mgSrtA variant can...

...each other very much undergo recombination, two smaller plasmids are formed. Viral vectors are prone to... the 5’ and 3’ LTR regions, it splits into two smaller plasmids. The ~3.7 kb recombined vector backbone...can improve growth and DNA yield. Test multiple, small colonies After streaking LB agar plates, you may...may notice that there are both large and small colonies. Bacteria containing the recombined product tend...tend to grow faster, so we recommend picking the smaller colonies which are more likely to carry the full...whereas the round tan colonies that look similar to normal E. coli cultures contain the recombined vector ...nicked, linear, and supercoiled. Here we find a smaller, recombined plasmid around 1.5 kb. When cut with...