...pAAV-FLEX-EGFPL10a EF1a-driven and Cre-dependent EGFP-tagged ribosomal L10a 5, rg* Heintz , Nectow , Schmidt Neurophysiology...

...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...

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

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

... 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 ...

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

... 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...

...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,...

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

...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...