...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....

...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...

...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...

...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...

...biosensor Keima is a mammalian dual-emission autophagy biosensor. Its resistance to lysosomal proteases is key...targeted to the vacuole in yeast or the lysosome in mammalian cells. This decrease in pH quenches SEP but not...allows for clear distinction between vacuolar/lysosomal and cytoplasmic Rosella. When fused with a mitochondria-targeting...remember that because Keima’s fluorescent relies on lysosomal acidity, this biosensor can’t be used with fixed...phosphatidylethanolamine (PE) and recruited to autophagasomal membranes, which leads to its degradation by...Tamotsu Yoshimori, and Beth Levine. "Methods in mammalian autophagy research." Cell 140.3 (2010): 313-326...measurements of macroautophagy and mitophagy in mammalian cells using a single fluorescent biosensor." Scientific...

...protein that enhances prime editing in mammalian cells: the small RNA-binding exonuclease protection factor...specialized prime editors (PEs). PE6a and PE6b are small prime editors with RT domains derived from the E...pombe Tf1 retrotransposon RT, respectively. Their small size comes at the cost of improved efficiency, although...and M-MLV RT enzymes, respectively, to obtain PEs small enough to deliver with AAVs but still efficient ...improves editing efficiencies compared to PEmax with normal pegRNAs, but combining PE7 with epegRNAs resulted... ✓ Evolved reverse transcriptase domains Smaller size and/or improved efficiency         ...used to rescue a genetic disease phenotype in an animal model, which was made possible through their use...

...relatively small and easy to work with, making them the go-to choice for studying mammalian biology. Similar...from S. pyogenes and has the advantage of being smaller (Zhang et al., 2016). For editing the genome, the...be electroporated into the oviduct of pregnant females, a technique called Genome editing via Oviductal...either an oocyte or into the oviduct of pregnant females (Yoon et al., 2018, Mizuno et al., 2018). Alternatively...for generating mutations by NHEJ and generating small mutations with HDR, when it comes to larger scale... SL, Kutny PM, Zhang Y, Cheng AW, Jillette NL, Malhotra A, Geurts AM, Chen Y-G, Wang H (2015) Efficient...srep32565 Resources on the Addgene Blog CRISPR 101: Mammalian Expression Systems and Delivery Methods Learn ...

... are much smaller and would be ideal for such AAV packaging, but their activity in mammalian systems has...packaged into AAV particles for delivery into mammalian cells, especially when fused to effector proteins...this problem. A miniature CRISPR-Cas system for mammalian use Stanley Qi’s lab has developed a miniature...activation, base editing, and genome editing in mammalian cells. The “V4” version (named CasMINI) had the...noted that Cas12f1 had an extra-long gRNA for its small size. They were able to increase activity and decrease...10kb but it is also capable of coding concurrent small insertions to allow in-frame deletions and introduce...program large genomic deletions with or without small insertions. But, instead, PEDAR couples this deletion...

...Polyclonal antibodies are generated by injecting an animal with an immunogen, then isolating and purifying....com. To generate polyclonal antibodies, an animal (such as a rabbit or a goat) is injected with an...response starts to wane about a month later, the animal is given additional booster immunizations every...reached - typically within 2-4 months. After the animal has the desired level of antibodies in its bloodstream...retrieved first by drawing blood from the immunized animal. The red blood cells and the serum are separated...different affinities for IgG subtypes from different animals so it’s important to choose the reagent that works...Only up to 5% of the IgG antibodies produced by an animal are specific for the immunogen. A different type...

... with in vivo experiments to determine the optimal/minimal injection titer and volume needed to achieve... now be able to make smaller adjustments to a set of injections into your animal model. For example, in... injection titers and volumes to determine the optimal injection parameters needed to transduce your cells...weeks allows the researcher to determine when the optimal time to conduct an experiment begins. This can ...the DIO/FLEx vector Once you have determined the optimal injection parameters for the serotype that best..., except now you will already have a starting “optimal” injection volume and titer to begin with. Now ...lab (Lavin, TK. 2020) it was determined that the optimal titer to prevent off-target expression of the sensitive...

...to insert at these sites.  Transgenic animals Transgenic animals are often generated by directly injecting... has a close-to-random integration profile in mammalian genomes. SB is active in vertebrates and integrates... well as in vivo in yeast, plant, insect, and mammalian cells, including human cells. piggyBac is biased...transposase (hyPB) is ~10-fold more active in mammalian cells compared to the codon-optimized wild-type...)TTATAA(G/C)TNA. Tol2 can deliver 10-11 kB to mammalian cells without decreased efficiency, with a maximum...human cells than piggyBac and Sleeping Beauty. Minimal Tol2 or miniTol2 is a truncated version of the ...Tol2 have all been used to generate transgenic animals, including zebrafish, mice, rats, and rabbits (...

...a blastocyst which is then implanted into a female animal for gestation. Nucleases that induce double-...are a common model organism used to understand mammalian traits and genetically engineered mouse models... during the DNA repair process. The addition of small molecules that promote homologous recombination ...microinjected into a zygote, which is then implanted into a female mouse for gestation. All three can introduce multiple...homology to the site of interest or create mosaic animals where not all cells in the blastocyst were successfully...identical mice. Thus, scientists must genotype each animal to identify the ones with the desired genotype....gene expression to be controlled through a drug or small molecule that regulates Cre expression. The most...

...include polymerase chain reaction (PCR), cloning, isothermal amplification, reverse transcription of RNA into...

.... 2014. Reprogramming adipose tissue-derived mesenchymal stem cells into pluripotent stem cells by a mutant...

...and the best practice to determine optimal DOL is through several small-batch experimental labeling. Size... century, it can be overwhelming to select the optimal dye or fluorescent ligand when designing an experiment...enzyme variant tag that covalently reacts with a small substrate ligand motif attached to a fluorophore...response to a specific stimuli, which leaves only a small volume of fluorophores in an excited state, effectively...to protein labeling and analysis, dyes should be small enough not to hinder protein function and should...affinity and specificity to nucleic acids, with minimal background staining. Additionally, cell permeability...2022). Caveat fluorophore: an insiders’ guide to small-molecule fluorescent labels. In Nature Methods (...

... Protein tags are usually smallish peptides incorporated into a translated protein. As depicted in the...point in crystallizing it or testing its function. Small ubiquitin-related modifier (SUMO) can help with ...with folding and stabilization, as can maltose-binding protein (MBP). Overexpression can also lead to insolubility... issues, but scientists may also choose to add smaller proteins, such as Thioredoxin A (TrxA) that improve...purification An affinity tag, generally a relatively small sequence of amino acids, is basically a molecular...simplest affinity tag is the polyhistidine (His) tag. Small and unlikely to affect function, His-tagged proteins... His HHHHHH  0.8 Affinity and Purification Very small size, rarely affects function S-tag KETAAAKFERQHMDS...

...understand how to use it. TLDR: This is a versatile mammalian expression vector that you can use to express ...express both SpCas9 and your gRNA in a variety of mammalian cell types. Purpose  SpCas9 This plasmid expresses...that the Zhang lab has tested in a variety of mammalian cell lines. Cas9, of course, is the key component...backbone can be used to transfect a variety of mammalian cells and can be modified for viral trasduction...by western blot. Overall, this is a versatile mammalian expression vector that you can use to express ...SpCas9 and your gRNA of interest in a variety of mammalian cell lines. Alternative forms of this vector contain...Some Common Ways to Deliver CRISPR Plasmids to Mammalian Cells Resources on the Addgene Website Visit...

...candidate in the Yang Lab, Qi Li, a lecturer at Sichuan Normal University, Jieze Zhang, an intern in the Yang ...recently, we’ve developed a new system “Multicopy chromosomal integration using CRISPR-associated transposases...great for experiments that require only single chromosomal integration (ex: genome editing), our MUCICAT...transposase system, INTEGRATE, is best for multicopy chromosomal integration (ex: for protein expression) (Zhang...Zhang et al., 2020). Multicopy chromosomal integration for gene expression is an alternative versus plasmid-based...advantages: Fast and site-specific multicopy chromosomal integration of cargo without using screening ...Molecular Plant Sciences in 2019. She joined Sichuan Normal University and became a lecturer in the College...

...different animals. Check out the Addgene website for plasmids that contain tinman or the mammalian homolog...character but rather to an extremely important gene in animal development. To understand where sonic hedgehog...that flies with non-functional hedgehog exhibit abnormal segmentation during embryogenesis. Typically, ...hedgehog (Nusslein-Volhard and Wiechaus, 1980). The mammalian hedgehog protein, sonic hedgehog (SHH) was named...pathway, which is a key regulator of all bilateral animal development. SHH plays a key role in proper limb...non-viral system to integrate particular genes into mammalian genomes and easily disrupt gene expression to ...arrest gene required for spermatogenesis One of our smaller conference rooms is named Cookie Monster. cookie...

...example, the visualization of chromosomal translocations or chromosomal shattering (chromothripsis). As...could then be used to examine both inter- and intrachromosomal dynamics, and how they change in response ...turned their attention to mapping pairs of intrachromosomal loci. The technique successfully resolved ...the degree of chromatin compaction even for this small distance! To the authors’ knowledge, this work represents... represents the first mapping of intrachromosomal loci, a major benchmark in characterizing the 4D nucleome...next! References Multicolor CRISPR labeling of chromosomal loci in human cells. Ma H, Naseri A, Reyes-Gutierrez...

... the identification of unknown GEFs for small GTPases." Small GTPases 7.2 (2016): 93-106. FREQ-Seq To...facilitate gene expression in bacteria, yeast, mammalian cells, insects, and even Xenopus oocytes. The ...promoters followed by leader sequences (encoding small leader peptides) downstream of which you can clone...Synthetic Inverters from Zinc Finger Proteins and Small RNAs." PloS one 11.2 (2016): e0149483. Rogers,...can be generated in a cost-effective manner with minimal bias. The FREQ Seq kit has been used in microbiology...2013): 5373-5383. Walkiewicz, Katarzyna, et al. "Small changes in enzyme function can lead to surprisingly...segments of choice into either an E. coli or a mammalian expression vector in a one-pot reaction. Using...