... yeast two-hybrid assay are not optimal for studying native mammalian protein interactions, whereas others...is needed to assess its function in mammalian cells or other animal models, but this technique has laid...p2A-ferritin-p2A-mCherry Mammalian Expression 74308 pcDNA3.0-Magneto2.0-p2A-mCherry Mammalian Expression 74334...sheared genomic libraries are cloned downstream of a minimal promoter sequence. Should one of these random stretches...it will activate its own transcription from the minimal promoter and its strength can be determined from...RAS Pathway Clone Collection 2.0 Ras proteins are small GTPases involved in cell signaling pathways that...been difficult to target therapeutically due in no small part to the central role Ras proteins play in cell...

...expression in neuronal cells. U6 small RNA expression shRNA From the human U6 small nuclear promoter Constitutive...expression) you need to use an RNAP II promoter, whereas small RNAs (such as shRNA) are transcribed from the RNAP...Similarly, the various eukaryotic cell types (mammalian, yeast, plants, etc) require unique promoters ...leakiness" of transcription may occur. This is normally not a big issue for cells, but it may confound...listing some of the most common bacterial and mammalian promoters. These lists is by no means exhaustive...considerations CMV General expression mRNA Strong mammalian expression promoter from the human cytomegalovirus... types. EF1a General expression mRNA Strong mammalian expression from human elongation factor 1 alpha...

... by the heat. Isolating male and female flies Differentiating male and female flies is essential for setting...of their abdomens, males have black coloration whereas females do not. Also, males have sex combs on their...is important to isolate females from males while they are still virgins. Females begin mating ~6 hours ...trying to get males to recombine two mutations onto the same chromosome. You should use females for that. ...their forelegs which are black, bristly hairs. Females do not have sex combs. To make genetic crosses ... bodies. This is the ideal stage to select for females because you can be certain that they are too young...fourth chromosomes, you are a very unlucky person. Animals are diploid except for their germ cells (sets of...

...screening 768 F1 animals). Recovering rare editing events in a sea of unedited animals struck me as a “...from 11-100% of F1 animals carrying precise knock-ins, and homozygous knock-in animals can be obtained in... in eight days. The only animals on a plate, other than the parental animal, are rescued progeny, which...for any selective marker to remain in the edited animal. Development of CRISPR/Cas9 editing strategies...must be removed, which can be done by isolating animals with particular visible phenotypes. These approaches...conditional-lethal mutation to identify edits with minimal screening effort. Selecting for repair of a lethal...unedited “haystack”, facilitating recovery of edited animals. Figure 1: In the background are 504 restriction...

...scientist Twitter users are male and 40% female, for a ratio of female to male = 0.629 (the closer this ...closer males and females are to parity - the smaller the number, the more it leans towards males). This...dataset. 3. 60% of scientists on Twitter are male and 40% are female Figure 2. Gender Ratio of Scientists on...is more skewed towards men than the ratio of female to male Internet users that use Twitter (0.875), but...but less skewed than the ratio of female to male scientific authorship in the United States (0.428). 4...

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

...induced by the addition of a small molecule, and protein levels can return to normal once the drug has been ... expressed beyond the tag and small molecule. It does work in mammalian cells, mice, and even yeast, though...conditions (typically the addition of an exogenous small molecule). Degron tags can range in size from several...degrons and some of the nuances to each one.   SMASh Small molecule-assisted shutoff (SMASh) functions on the...itself when you want the protein around, leaving a minimal additional sequence (7 kDa). This system also requires...are 1) a SMASh-tagged protein and 2) asunaprevir (small molecule). dTAG The degradation tag (dTAG) system...selective degrader. The novel degrader, dTAG13, is a small molecule which induces dimerization of the FKPB12F36V...

... but it’s really just formaldehyde. Formalin: You may also come across formalin, which refers to a saturated...cause some confusion. Paraformaldehyde: Many IF protocols call for paraformaldehyde (PFA) as the fixative...saturated solution of formaldehyde (saturated being 37%). Formalin solutions often contain methanol as ...order to slow the polymerization of formaldehyde.  Note: Formaldehyde in solution will naturally polymerize... maintaining cellular and tissue morphology. Formaldehyde is the most commonly used cross-linker for IF...fixative. PFA is a polymer of formaldehyde that, on its own, is not a fixative. However, when PFA is dissolved...solution and heated, it depolymerizes, producing formaldehyde. Often people will call the resulting solution...

...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 ...

...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...

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

...the human erythrocyte by the blood stage of the malaria parasite, P. falciparum . Please see the following...

...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...

... 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...

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