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...Goedhart and Marieke Mastop from the Section of Molecular Cytology and Van Leeuwenhoek Centre for Advanced...separate proteins based on size. Whether higher molecular weight complexes are detected will depend on the...spectroscopic methods (based on fluorescence polarization) can be used to detect homodimerization. However...interact or oligomerize in cells? This question is largely unanswered. Ideally, fluorescent proteins should...fluorescent proteins approach this ideal situation, cellular assays have been proposed. Early efforts tested...non-dimerizing if its name is preceded by an 'm' to declare that it is monomeric. The claim that a fluorescent...Goedhart is an assistant professor at the Section of Molecular Cytology and Van Leeuwenhoek Centre for Advanced...

...that you can make a very large number of regulators. That basically means larger genetic circuits that could...way. In one of his latest papers, published in Molecular Systems Biology in November, Voigt and Alex Nielsen... could conceivably be as large as natural regulatory networks. Addgene: What kinds of things could we ...we do as genetic circuits become larger and more complex? Voigt: If you look at all the products of biology... of hurdles we haven’t yet overcome. For this particular paper, there actually were not many hurdles. ...switch-like at all. It’s also difficult to build larger circuits with many guide RNAs connected because...repressors, activators. We’ve been putting together very large toolboxes and making them available. We’ll also ...

... cells (e.g., a neuron subtype) and specific subcellular compartments for measurements. All of the sensors...whereas non-neuronal systems tend to have slower, but larger, calcium changes. The GCaMP6 family of sensors ...Localization The calcium concentrations in different subcellular compartments can vary greatly. Additionally, ...example, calcium in the ER lumen reaches the sub-millimolar range, much higher than in the cytosol. Thus,... aims and requirements of in vivo studies. A particularly useful sensor for neuroscientists is CaMPARI... violet light and (2) exposure to increased intracellular calcium. It has been applied to mark active ...Whether you’re studying the brain, muscles, or intracellular landscape, remember: Keep Ca+2lm and Ca+2rry...

...studying synaptic connections and physiology is particularly challenging as they are great in number, generally...the target gene’s native locus, but introducing large genetic changes in C. elegans with CRISPR can be...(ssODN). So how can you use CRISPR to introduce large FP genes into C. elegans? By using a split FP approach...express wrmScarlet1-10 unattached to any other cellular protein. They used CRISPR with a ssODN template...-wrmScarlet labeling proteins with different subcellular locations. Image from Goudeau et al., 2020. ...difficult to observe the cell cycle in vivo, particularly if the cells are migratory or in deep tissues...plant cells. Its small size offers advantages for cellular delivery. Get the plasmids at Addgene Sc++ is...

...patients is not surprising. However, the molecular and cellular mechanisms behind the immune defects in...Glycoimmunology group based at the Research Unit on Applied Molecular Biosciences (UCIBIO) focused on understanding ...proteins assemble into functional multi-subunit macromolecular complexes, but a serendipitous discovery led...guanidine hydrochloride, as a potential treatment for a large number of rare genetic diseases. Spontaneous mutations...causing release of acetylcholine. Given its unique molecular biology and previous approval by the FDA, GdnHCl...that cause rare diseases. They now plan to test a large number of other disease-linked Arg-mutant proteins...human cells in order to determine the specific molecular features that allow such “chemical rescue” in ...

...inserting large tags into a precise location in the genome is still inefficient, particularly in human ...true in the case of protein tagging to elucidate cellular structures. Overexpression methods flood the cell... we’ve tagged are widely recognized markers of cellular structures, we provide guidance on how to make...fact. Later, when we transfect the cells, these large regions of homology enable us to effectively utilize...-terminus of the gene of interest. To get the molecular components of CRISPR/Cas9 (Cas9 protein and the...and design described above can be used to create similar donor plasmids for introducing tags into your gene...

... still needs to be taken when selecting which particular AAVs to use for your research. Below I’ll provide...preferential tropism for different brain regions is largely due the ability of different serotypes to infect... you should try to find the best AAV for your particular experiment! Traveling from the Original Location... and anterograde transport, but they also have similar transport characteristics (velocities of movement...is fine and a researcher should aim to inject a larger amount of vector using a serotype with strong CNS...receptors (i.e. chemogenetics vs. optogenetics) I’m particularly interested in using chemogenetic and optogenetic...vectors, insertional mutagenesis, and cancer." Molecular Therapy 15.10 (2007): 1740-1743. PubMed PMID: ...

...recombination leading to helper virus production." Molecular and Cellular Biology 6.8 (1986): 2895-2902. PubMed PMID...vector system. Viral vectors have been increasingly popular in fundamental and applied research since their...un-transfectable cells, labs use viral vectors and particularly retroviral vectors because they target a broader...was obvious that its safety was a disadvantage particularly for clinical development. In addition, MoMLV... the env gene (packaging plasmid), and 3) the vesicular stomatitis virus G protein (VSV-G) that improves...improves the stability and broadens the cellular tropism of the viral particles produced. However, in this... cell from the clinic. The 2nd generation is similar to the first one with the exception that the HIV...

...understand the intricacies of molecular pathways. These screens have gotten larger, easier, and more comprehensive...Forward genetics screens are a valuable part of the molecular biology toolbox to identify new target genes for...lentiviral-based CRISPR libraries available at Addgene. The large packaging capacity and stable expression of lentiviral...may be too constraining for some experiments, particularly in vivo ones. In those cases, AAV vector screens...your CRISPR screen to a high degree. This is particularly helpful when investigating tissue-specific diseases...relies mainly on episomal expression. Episomes are circular pieces of DNA that reside outside a host cell’... vivo CRISPR screens. AAV vectors have played a large role in these advancements, due to their many benefits...

...2–3 ml Skill required: intermediate Intramuscular Intramuscular (IM) injections occur, as the name suggests... surrounding organs in the abdomen. They are a popular choice for administering different drugs and viral...injection site is straightforward, as it targets large muscle groups (usually the thigh), though it is ...theoretically be performed anywhere on the body, somewhere large, flat, and easily accessible (like the back) is ...Intranasal instillation Intranasal instillation is very similar to its intratracheal counterpart, as both procedures...Peritoneal cavity 25–27 < 2–3 mL Intermediate Intramuscular Mechanical restrainer Thigh 25–27 < 0.02–0.05...Mouse Modeling Part 1 Mouse Modeling Part 2 Five Popular Model Organisms Additional resources on Addgene.org...