...activators were constructed, with VP48 containing 3x minimal VP16 domains, VP64 with 4, VP96 with 6, and VP160...

...nor the small molecule are strongly fluorescent but the binding of an aptamer to its target small molecule...Fluorogenic RNA Mango aptamers for imaging small non-coding RNAs in mammalian cells." Nature communications 9.1...nucleic acid oligos that bind with high affinity to small molecules to induce their fluorescence. Until a ...molecule activates the fluorescence of the small molecule. Fluorescent RNA aptamers are used to track cellular...encoding aptamer-tagged RNA and later introducing the small molecule into the cell. The fluorescence that is...their derivatives are aptamers able to bind to a small molecule derivative of GFP called 3,5-difluoro-4...highly unstable in vivo due to poor folding and thermal instability at 37°C (Ouellet et al., 2016) and ...

...Wright Lab plasmids EXPRESs is a collection of mammalian expression plasmids that encode the extracellular...

...diseases, animals have reduced anxiety levels and a higher tendency for risk taking. These animals also show...time the animal spends in the passive mode, the stronger is the depressive component of the animal behavior...and quantify locomotion, scientists monitor an animal’s movement in an open field box or a circular corridor... other aspects may be involved. For example, an animal moving around to explore the environment has a ...motivation (and brain areas associated) compared to an animal running to escape a threat. Locomotion can thus... study certain forms of drug addiction. When an animal has been exposed to morphine or other stimulating... to the NAc and decreased the locomotion of the animal. This confirms the previously suggested inhibitory...

...fluorescent dye or HRP via maleimide-labeling. Maleimide-labeling occurs when a maleimide group forms a covalent...found in cysteine. Maleimide-conjugated HRP can be found here and several maleimide-conjugated dyes are...broken,” their production does require continued animal sacrifice. Could there be an alternative method...comparison As the name suggests, nanobodies are smaller antibodies.They are derived from an unusual type...bacteria, which means their production requires animal sacrifice and precludes genetic engineering of ...Commercially available Not recombinant; Require animal sacrifice Poor tissue penetration Less resolution...expensive Nanobodies   Recombinant; do not require animal sacrifice Good tissue penetration Can be labeled...

... CRISPR systems Smaller Cas proteins SaCas9 isn't the only CRISPR enzyme that’s small enough to package... and its gRNAs into one AAV, Cas9 must be made smaller. Previous attempts to “shrink” Cas9 include the...than its wild-type counterpart.  In search of a smaller, but equally potent Cas9 protein, the Zhang lab...1053 amino acids) displayed cleavage activity in mammalian cells. SaCas9 produced indels at a similar efficiency...post-injection. Livers from these mice were histologically normal and liver injury markers were not increased compared...Cas9 from Campylobacter jejuni (CjCas9) is the smallest Cas9 ortholog characterized to date. Kim et al...disease-causing loci in mouse liver.  Two other smaller Cas proteins include Cas12b and CasX. While there...

...natural mammalian plasmids, mammalian vectors are even less similar to bacterial ones. The mammalian analogue...vector remains episomal, meaning it stays separate from the genome. One option for episomal vectors is to...bacteria first. In fact, most yeast vectors and many mammalian vectors are designed to shuttle between species...Alternatively, some slow-growing cells, particularly mammalian cells like neurons, may not replicate during the...yeast species and the goal of the experiment Mammalian cells At least one of the following: to stably...across kingdoms by targeting a shared aspect of ribosomal function. A puromycin resistance gene on your ...synthesis machinery, and the cells are switched to minimal media, allowing cells that contain the vector to...

... knockouts. Mammalian and fly applications MoClo was initially developed for non-mammalian systems, but...in bacterial and cell-free expression systems, mammalian cells, yeast, flies, and even plants! In this ...range of parts, including copy number machinery, chromosomal integration modules, and many more of the usual...conditional gene expression, multiplexed CRISPR, and chromosomal integration of larger expression cassettes (Shaw...addition to additional elements for S. cerevisiae chromosomal integration and gene expression, the system includes...but it is not confined to that arena! The Mammalian Toolkit was engineered specifically for the types ...types of expression and integration needed in mammalian cell types, and comes with a large library of common...

...while mAbs generally require mammalian expression systems. scFvs are also small enough to be screened for...genes from B cells of immunized animals, but it's possible to avoid animal use by using semi-synthetic or...al., 1988; Huston et al., 1988). scFvs are the smallest unit of an antibody molecule that can bind antigen...scFv’s advantages are rooted in their size. Being smaller means scFvs are easier and cheaper to make because...Bradbury et al., 2011). In vitro selection avoids animal immunization and also allows for generations of...or self antigens, and toxins that are lethal to animals.  In the clinic, scFvs’ size also provides advantages...and a higher likelihood to aggregate due to their small size (Bates and Power., 2019). Their rapid clearance...

...spread of cancer cells. Besides these stromal cell modifications, stromal cells are also constantly communicating...microenvironment’s malignancy but also incite different organs to become malignant. Exosomes also contain...on the conversion of non-malignant forms of cancer to invasive and malignant forms and noticed that myoepithelial...inflammatory, angiogenic vascular, endothelial, and stromal cells - a rich soup of life encircling the tumor...that when there’s less caveolin 1, there’s less malignancy because there’s less proliferation. However, ...caveolin 1 in breast cancer also induces changes of stromal cells in the microenvironment. This results in ...exosomes are tools of communication and provide malignancy to the microenvironment itself. They deliver ...

... mind before choosing a system. Mammalian expression systems Mammalian cells are an ideal system for the...protein production and purification. These include mammalian, insect, bacterial, plant, yeast and cell free... the expression of mammalian proteins that require multiple post-translation modifications for proper ...protein function. Most DNA constructs designed for mammalian expression utilize viral promoters (SV40, CMV,...RSV) for robust expression post-transfection. Mammalian systems can express proteins both transiently ...protein yields if transfection is successful. Some mammalian systems also allow for control over when a protein...high concentrations. Despite their advantages, mammalian expressions systems do require demanding cell ...

...DNA sample being sequenced must be sheared into smaller fragments that are then barcoded for identification...library molecules for each sample remain highly normalized across a wide range of input DNA concentrations...explained more fully in the animated Fig. 1 below. Normalization is a fundamental and often understated problem...sufficient coverage of all samples. The root of the normalization problem often arises from the fact that large...issues, researchers usually include a separate normalization step where individual input DNA samples are ... to library prep. Even with this “pre-prep” normalization, it is also often the case that individual libraries...samples will then also need to be quantified and re-normalized in order to be combined with other samples in...

...Libraries gRNAs Empty gRNA Vectors gRNAs By Species Mammalian Bacteria Drosophila Plant C. elegans Yeast Zebrafish...

...thoroughly between animals as olfactory stimuli that were left behind from a preceding animal (ex: urine) can...Interaction should be minimal and strictly controlled; optional handling animals Experiment Behavioral...testing apparatus. General stress levels of the test animal may also be reduced by daily handling for a few... can be distracting. If it is not cleaned, animals tested towards the end of an experiment series would...Variability source Standardization parameters Animal Strain, age, sex, housing conditions (individually...method uses Cre recombinase on transgenic flox animals to introduce modifications at the DNA level in ...for intracerebral injections. AAV particles are smaller and may be preferable if wider diffusion is required...

...their limitations for peering right through living animals to see their organs – a liver or brain, say, or...electromagnetic spectrum – the spectral region in which mammalian tissues are nearly transparent. As Verkhusha explained...explained it to me, the transparency window of mammalian tissues is set by the properties of hemoglobin...wavelengths between 650 nm and 900 nm will pass through animal and human tissues largely unimpeded. The challenge...proteins allowed a signal-to-background ratio in mammalian tissues 20-fold greater than any fluorescent protein... fluoresce outside the transparency window of mammalian tissues,” Verkhusha said. “We developed near-infrared...photoacoustic contrast agents for two-color imaging in animals. The approach relies on ultrasound waves as opposed...

...powerful tool to modify mammalian cells. Multicolor CRISPR labeling chromosomal loci in genome Thoru Pederson...righlty deserve, but, in this post, we'll provide a small sampling of the many amazing new plasmid tools that...regulation has roles in many cellular functions, from endosomal trafficking to signaling pathways to the cell ...for light-mediated subcellular localization in mammalian cell culture and reversible control of signalling... *False positives can occur in this system if a small contaminating PCR product formed from primer dimers...translation occurs in both live cells and whole animals. The system utilizes the bulldozer-like quality...dynamic interactions between intra- and inter-chromosomal domains. The authors anticipate that this tool...

...manipulate the organism they study (unless if you count normal human interaction for all of us studying human ...gene function and phenotypes in diverse plants, animals, microbes, viruses, and fungi for which these methods...Creating a productive, new scientific community is no small task. The EDGE program solicitation requires funded...materials. Many groups publicized workshops on their organismal systems for scientists and educational programs...organisms for genotype to phenotype studies New organismal systems described at this meeting were extraordinarily...including genetic mechanisms that control who a female wants to mate with and if she decides to raise ... and maintenance that had the audience gasping. Animal husbandry of diverse species is a real bottleneck...

...said, normalizing to total protein expression is preferable, and more accurate, than normalizing to ubiquitously... a Western Blot post to make sure you have the optimal gel for your experiment.   Ladders If you need ...common (non-technical) issues with transferring are smaller proteins going completely through the membrane ...proteins: try using a 0.2 µm (psi) membrane for small proteins (< 15 kDa) and a 0.45 µm (p) membrane for...step to take. If you’re not sure if you need a smaller pore on your membrane, run a double-membrane transfer...the system cool, as can lower voltages.  If your small protein is over-transferring, try using the faster...faster semi-dry transfer, which works better for small and medium proteins. If your proteins are compatible...

... to male stem cells. Nor did they detect important differences in function between male and female cells...stem cells in mice. They appropriately used male and female mice (using both sexes in basic research is...such case study looks at the effects of using all female cells and tissues, specifically in stem cell therapies...is excellent design), but they used all female stem cells—this was an unconscious and arbitrary decision...considering the sex of the stem cells was that their male mice died—and they didn’t know why. Eventually, ... wanting to consider sex is cost. Especially in animal research, they worry about needing more study subjects...is that cost at this early stage of research is minimal compared to that of human clinical trials. And,...

...If your protein of interest is small, you may want to consider a smaller pore size (0.2 µm). Conversely...protein capacity, allowing you to optimize for smaller proteins or lower fluorescent backgrounds. However...but it is not as efficient. It is recommended for small to medium proteins. Finally, there are dry/electroblot...higher specific activity, slightly lower cost, and smaller size. However, HRP is not compatible with sodium... provide a relative, not absolute, readout. Normalization Overall protein expression varies from sample...compare one sample to another, you’ll need to normalize your protein of interest to overall protein expression...changed due to ‌experimental conditions. You then normalize your protein of interest to total protein expression...

...your opsin through a small piece of fiber optic cable implanted into the animal’s skull (right). The fiber... using stereotaxic surgery and cemented to the animal’s skull using dental cement (a similar procedure...subsequent stimulation, the rate of movement of the animal significantly increases. Before you get your experiment...caveat is that one laser can stimulate only a single animal during the course of an experiment. There are a... control one or two lasers are also available. Optimal stimulation parameters must also be determined ...behavioral optogenetics is performed in freely moving animals, the fiber optic patch cable must be the appropriate...that it becomes tangled. Furthermore, to give the animal the full range of motion, a commutator allows the...

... 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...

...Autophagosomes 101784 SLC25A17-mEGFP AICSDP-27 mEGFP Peroxisomal membrane protein PMP34 Peroxisomes 101785 TUBA1B-mTagRFP-T...

...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...

...Uncovering the role of p53 splice variants in human malignancy: a clinical perspective. Surget S, Khoury MP,...