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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 ...

...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...

...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...

... 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...

... 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...

...protein, or cellular structure and a particular biological event (trafficking to a subcellular location,... a microscope, fractionate samples to isolate particular organelles and their contents, or perform in ...result of chromophore oxidation. Some of the more popular timers (whether as part of protein fusions or expressed...separate proteins should be useful for monitoring cellular events occurring at varied time scales.  Table...lysosome-associated membrane protein, and following its subcellular localization overtime (Subach et al.) Monitor...developed monomeric fluorescent timer protein." Molecular biology of the cell 21.1 (2010): 87-94. PubMed...timers that change color from blue to red report on cellular trafficking." Nature chemical biology 5.2 (2009...

...Cellular communication and function depend on potential energy. Cellular activities like action potentials...mentioned, cellular voltage is measured at the cell membrane, and this voltage is particularly physiologically...heart issues stem from the heart beating at an irregular or insufficient level. The heartbeat – a muscle... change across the membrane of cardiac cells. Similarly, neuronal signaling is carried out by action potentials...quenching of the fluor via FRET. This system is particularly powerful for in vivo imaging especially since... This specific example is attractive due to the large number of sheer molecules and signal you can generate...membrane, otherwise they aren’t all that useful. The cellular localization component is typically handled by...

...respectively, quickly became popular, and have maintained that popularity through the years.  Libraries... and requesting each year (Figure 2). We did a similar analysis with CRISPR pooled libraries, although...nucleases, the original CRISPR tool, remain the most popular deposited and distributed CRISPR plasmids. But ...were knockout libraries, and they remain the most popular with our requestors. However, we can again see ...Figure 3), we saw that once CRISPR plasmids become popular, they continue to be requested through the years... deposited, you can see that from 2013 and on, popular CRISPR plasmids seem to never truly go out of style... in Figure 3, indicates that the usefulness of popular CRISPR tools is both enduring and rapidly recognized...

...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...

...designed to hold large amounts of data. Although it may be useful to selectively bring particular items into... permissions to view and edit to particular groups and/or particular individuals, the issue of third-party...the University of Goettingen, and several other large research institutions. The following graphic depicts...small fraction of all data directly relevant to a particular experiment, series of experiments, or project...Integrations with additional tools are added on a regular basis, so the ecosystem of related tools is always...capture the conversations and associate them with a particular experiment or project thus significantly enhances... Resources on Addgene.org Find protocols of molecular biology techniques Find plasmids at Addgene Make...

...CRISPR, is turning potential into impact. In molecular and cellular biology, this happens through the development...this information to identify a large variety of related bacteria with similar Cas systems. Some of these ...exclusively on this space — developing tools for cellular targeting. Lately, they’ve been working on two...of Cas7-11 were reported as up to 85%, broadly similar to shRNA and Cas13 systems in a range of mammalian...fused integrase would then integrate into with a large DNA insert – or Programmable Addition via Site-specific...programmed for new genes. In comparison with other large insert techniques like HITI or HDR, which require... and adenovirus templates. The system, which is larger than Cas9, can also be used with mRNA and a synthetic...

... cell types depending on their serotype, are particularly challenging. Their efficacy in any given system...and to verify the quality of the prep. This was a large factor in Addgene’s decision to launch our viral...category, available from Addgene as of 2023. A large percentage of viral vector preps contain biosensors...were distributed in 2023 alone. You’ll also find a large number of requests for vectors in the “controls”... 4: A) Cumulative AAV preps for the seven most popular serotypes available from Addgene from 2016–2023...2023. B) AAV preps for the seven most popular serotypes distributed per year by Addgene from 2016–2023. ...in 2016, and it quickly became one of our most popular ready-to-use viral vectors, making up almost 10%...

...Replication and control of circular bacterial plasmids. Microbiology and Molecular Biology Reviews: MMBR, ...Plasmids 101 Posts Get practical molecular biology help with our Molecular Biology Reference Pages   ...maintaining a stable concentration of plasmid. Similar to replication in E. coli, regulation primarily...replication of the plasmid proceeds (and terminates) similarly to the bacterial chromosome. Since replication... thereby the overall replication of pSC101 (del Solar et al., 1998). If that’s not enough to keep replication...must fold into a “pseudoknot” held together by intramolecular base pairs for the protein to be translated...5213. https://doi.org/10.1093/emboj/17.17.5201 del Solar, G., Giraldo, R., Ruiz-Echevarría, M. J., Espinosa...

...using electrophoresis, typically capillary electrophoresis. Capillary electrophoresis reads a fluorescent...repetitive regions or for higher accuracy of a particular sample.  Long-read sequencing (“Third-generation...technology, but Oxford Nanopore is one of the most popular options. Nanopore sequencing deviates from the ...reads using the BBTools suite, and assembles circularized plasmid sequences via a combination of open-...novo assemblers, so if one fails to generate a circular plasmid sequence, it moves to the next. If all...constantly faces the challenge of keeping up with the large amount of QC analysis required every week. They ...sequencing Technologies. Current Protocols in Molecular Biology, 122(1). https://doi.org/10.1002/cpmb.59...

...distinguish between two similar types of HPV, HPV16 and HPV18, from human cells, at atomolar levels, within one...platforms to reliably detect nucleic acids at the atomolar level. The Zhang lab has adapted natural RNase...reporter enables SHERLOCK to detect DNA or RNA with atomolar sensitivity and single base pair mismatch specificity...be detected on commercial lateral flow strips, similar to pregnancy tests. With lateral flow strips, the...CRISPR Cas variant that can also cleave dsDNA similarly to Cas9 (Zetsche et al., 2015 ). Cas12a however...DETECTR. How does DETECTR work? DETECTR works similarly to SHERLOCK. Cas12a is targeted to a specific ...nucleic acids in a mixed population for an array of molecular and clinical diagnostic applications. The company...

...also be used to map molecular pathways and genetic interactions. Additionally, large scale CRISPR-based...guides directed towards that gene as there can be a large difference in guide potency. Thus, in cases where...oligo library synthesis. Prior to Cas9 activators, similar tools were made using other DNA binding proteins...makes it much cheaper to use Cas9 activators. This larger scale of activation allows for exploratory-based...given cell type or organism, have been used in a similar manner to Cas9 activators. However, these can be...gene, as, many times, the isoform(s) common to a particular cell type are unknown or not readily available...target effects are generally not regarded as being a large problem for Cas9 activators. This is believed to...