...-invasive gene delivery and optogenetic excitation. Excitation of neurons deeper in the brain may still...makes them light-responsive. When light shines on excitatory channelrhodopsin-expressing neurons, the channelrhodopsins...action potential (Figure 1). This light-induced excitation can take place in a tissue culture dish but can...high-intensity light for activation which requires implantation of fiber-optic cables into the brain. ...still require fiber-optic cannula implantation, but altering ChRgers so they can be activated with tissue-penetrating...

...articles. Then just start reading and following citations through the literature until you have all of the...they mean. Could there be any other possible interpretation of the results? Then think deeper about the...Is the data of sufficient quality (error bars, statistics, clarity in images etc)? Did they perform all...doesn’t mean that there are no mistakes or misinterpretations. After all, the science was performed, written... brim with reprints and papers with scribbled notations in the margins. If you prefer, printing and hand-writing... Department of Biological Sciences at Louisiana State University. She is a fan of photosynthesis and writes...

...Scientist positions often required a one-hour presentation about a prior research project. In total, the...notes after each interview.   Tailor your 1-hour presentation to your audience Start by asking the hiring ...hiring manager or recruiter what presentation format they require. Frequently they will ask you to present ...giving them sufficient background info. Use the presentation as an opportunity to demonstrate technical and... potential technical difficulties. Have your presentation on a flash drive and consider bringing your ...company will allow it. Before you start your presentation, introduce yourself to your audience. Pay...

...efficient. Informed by these mutations and previously described TadA mutations (Gaudelli et al., 2017), they...withstand antibiotic selection. As a result, 8 mutations in TadA could create all three edits and were ...placeholder for the number that identifies the mutation in the evolved TadA domain. Because ABE7.10 contains...therapy approaches can use base editors to correct mutations in stem cells and return the corrected cells back...tested ABE8.17-m and found that one base change mutation (ABE8.17-m+V106W) (Gaudelli et al., 2020) was ...

...elegans. To study the role of 3’UTRs, the Arizona State University (ASU) Biodesign institute developed a...polyadenylation for all protein coding mRNAs, and annotations extracted from other databases (Mangone et al...Dynamics Database (WDDD) The WDDD aims to acquire quantitative data on all the cell divisions of the C. elegans...methods provides unique opportunities to develop computational methods to better understand animal development...additional SapTrap plasmids used for introducing point mutations that was published in a microPublication (Schwartz... E. M. "SapTrap vectors for introducing point mutations with unc- 119 + selection." MicroPublication 1...

...you'll find in this post: Tools for labeling excitatory inhibitory synaptic proteins Fluorescent protein...corner New from the viral service Labeling excitatory and inhibitory synaptic proteins with FingRs ...developed AAV vectors to selectively visualize excitatory and inhibitory synapses in the brain.  ...scaffolding proteins PSD95 and gephyrin to label excitatory and inhibitory synapses, respectively. Once expressed... fluorescent proteins targeting inhibitory or excitatory synapses or that are Cre-inducible, researchers... development, regeneration, and certain disease states. However, it’s difficult to observe the cell cycle...

...replicate while maintaining the undifferentiated “stem” state and, Their potency or capability to differentiate... mostly from abnormal and/or supernumerary preimplantation embryos that are produced during in vitro fertilization...during ESC isolation. Low Chances of post-transplantation rejection when used therapeutically High Low...similar functional utility, iPSCs bypass the transplantation compatibility issues and ethical concerns of... used therapeutically, the chances of post-transplantation rejection are low. Further, as iPSC generation...embryonic stem cell lines from IVF embryos after preimplantation genetic diagnosis. Hum Reprod, 2008. 23(1):...

...challenges, and the flood of interview requests and visitation days can be, at times, very overwhelming. As ...applied to PhD programs in Biology around the United States in 2021, I know that this process only became even... to a school that requires its students to do rotations in different labs before selecting an advisor,...prospective student of such-and-such university State how you found their lab (usually through the university...name of the award you won for your conference presentation. It can help soothe your nerves and make sure...on opposite sides of the country had scheduled visitation days during my midterm exams. Using the online...

... track of your mutation. To maintain stable lines of flies carrying a specific mutation, Drosophila geneticists...contributed by mother and father). While some mutations can remain stable as homozygous alleles, others...you can stably maintain a heterozygote, and the mutation won’t drift away with each passing generation....journey of trying to get males to recombine two mutations onto the same chromosome. You should use females...whether any phenotypes have been observed from mutations in the gene, and whether any publications have...

...localization and transport between healthy and diseased states can also provide interesting insights into disease...analysis software produces quantifiable results and statistics that can be used to compare between different...Dunn et al. 2011 provides an in-depth review of quantitative colocalization analysis methods and useful image... image analysis software tools. Pitfalls, limitations, and specialized colocalization methods Fluorescent... very useful tools, but they are not without limitations and well-executed experiments require some consideration...Fluorescent Proteins. Ruusuvuori, Pekka, et al. "Quantitative analysis of dynamic association in live biological...

...in an open inactive state, a closed active state, and an artificially active state due to closure by FP...The FP is confined to a plane and generally only rotates in 360°, increasing the probability that two FPs... and Citrine) can be monomerized with an A206K mutation [2]. Addgene has many of these constructs and ...used to determine if a protein is monomeric is sedimentation equilibrium analytical ultracentrifugation [...

... (NHEJ), usually results in a loss-of-function mutation within the target gene. CRISPR can also be used...cells, with each cell or set of cells containing a mutation in a different gene. Libraries may be available...correspond to genes that confer drug resistance when mutated. Findings from this type of experiment can shed...requires the use of next-generation sequencing. Representation - Most libraries contain 3-6 gRNAs per target... gRNA within the population is key. Loss of representation due to enrichment or depletion of specific ...CRISPR screen. However, maintaining sufficient representation within your mutant population requires a massive...

...These insights are hard to appreciate without computational support. Indeed, rational sgRNA design is not...painstakingly assessed to something a bit more representative of the communities’ opinion. The trouble is...pool of CRISPR software embodies solutions that facilitate most experimental applications in CRISPR engineering...function can be found in their online tool’s documentation. Since Hsu et al., 2013, other tools have incorporated...target sites, off-target sites, scores and the statistics that go into calculating them. For this, some... hardware. This OpenCL dependency is a severe limitation, but the tool is fast enough that, if you’re ...Python script called SSFinder, but I find its implementation to be too slow for practical use. The R based...

...inspiration behind ExM, how it works, and some of the limitations to keep in mind whether you are planning your...your specific targets. What are some of the limitations?  A “super-resolution” technique that uses common...great, right? But there is always a catch - some limitation of the technique that users should be aware of...First, expansion requires fixed samples. This limitation may be the both most obvious and least flexible...ensure the expanded images are still a good representation of reality. Third, expansion results in dimmer...density (Mikhaylova, et al. 2015).  Despite these limitations, ExM is a valuable tool, especially when paired...

... Albert Einstein might have put it best when he stated, “the value of an education . . . is not the learning...Provost, then a new faculty member at Minnesota State (and now Professor of Chemistry and Biochemistry...Community (MCC) that seeks to reduce barriers to implementation. Funded by the NSF (with Co-PIs Jessica Bell...Professor and Chair of the Biology Department at Keene State College in New Hampshire. He mentors undergraduates... Kowalski, J., Hoops, G., Johnson, J. 2016. Implementation of a collaborative series of classroom-based...Scientific Elite: Nobel Laureates in the United States. Free Press. New York. Resources MDH CUREs Community...

...Agrobacterium tDNA insertion likely precipitated domestication of sweet potatoes (Kyndt et al., 2015)! It sounds...plants. If your cultures have been incubated at stationary phase for too long, the cells in your culture...affected by the selection eventually. Genotype putative T1 transformants: When your T1 is on soil and .... et al. (2015) ‘The genome of cultivated sweet potato contains Agrobacterium T-DNAs with expressed genes... the National Academy of Sciences of the United States of America. National Academy of Sciences, 112(18...

...development, sex determination, and anterior-posterior segmentation (Jennings and Ish-Horowicz, 2008). Fruit flies...flies that contain a specific loss-of-function mutation in groucho have thick tufts of sensory bristles...with non-functional hedgehog exhibit abnormal segmentation during embryogenesis. Typically, regularly spaced...embryo depending on its concentration. Genetic mutations in SHH cause severe health conditions such as ...reconstructed a transposase system based on the naturally mutated and non-functional transposase gene found in salmonid...Nüsslein-Volhard, Christiane, and Eric Wieschaus. "Mutations affecting segment number and polarity in Drosophila...

...screens Improved prime editor PE7 Antibodies to facilitate SARS-CoV-2 research New viral vector preps of...-024-07259-6. PMID: 38570691. Antibodies to facilitate SARS-CoV-2 research   By Ashley Waldron Ready-to-use...HaloTag, Figure 4), they determined that an A122D mutation in the rhodopsin domain conferred several new ...potentials of fast-spiking neurons. The same A-to-D mutation in the related voltage indicator Ace2N-mNeon also...-microglia cells.      Figure 5: A) Representative confocal images showing GFP expression from...System By Vaibhav Kawde To circumvent the payload limitations of traditional AAVs, the Becirovic Lab has developed...

...engineered by introducing Cas9-mediated frameshift mutations in the gene of interest. The genomic target for...for Cas9 and the introduction of this mutation can be any site early in the coding region of the gene ... experiments may require the introduction of a mutation at a specific site e.g., deleting the catalytic...modification at a defined amino acid to mimic a patient mutation or maybe to disrupt a catalytic residue. Good ...to either cytosine or adenosine deaminases to facilitate a single base pair edit. (Cytosine deaminases...of activators include the SunTag system which facilitates recruitment of multiple activation domains to...

...under this ruling, regardless of visa or fellowship status, in addition to certain staff scientists and those... salary was set at $47,476 and the date for implementation was set for Dec 1 2016. In order to be exempted...paid. Institutional salary policies are one indicatator of how salaries may changes due to the FLSA. ... The University of Notre Dame sets no minimum, stating that while they recommend NRSA levels, postdocs...not to be tracked. University of Buffalo and Ohio State both have minima of $30,000, and raising salaries...addition, both Brown and Brandeis universities have stated that they consider postdocs paid on stipends to...

...how many pups will contain a mutation and many of the mice with the mutation will be homozygous for it in.../Cas9 for gene editing has expanded since its adaptation for use in mammalian cells in 2012-2013. Researchers...region. Or you may even wish to generate a point mutation and need to focus your attention to that particular...will require you to house mice that may have no mutation, costing money and space. As a rule, screen all... et al. "One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome...

...Garret D., et al. "Excitatory transmission from the amygdala to nucleus accumbens facilitates reward seeking...to your field. Ultimately, your question should dictate your behavioral output. However, regardless of ...behavioral test in which your circuit of interest is putatively involved in regulating the behavioral output....2) there is some problem with the technical implementation of your optogenetics setup (viral expression...and anxiety along the dorsoventral axis of the dentate gyrus." Neuron 77.5 (2013): 955-968. PubMed PMID...

... with sub-optimal codon usage (Table 1) A few mutations are common to all or most expression strains to...levels including:  ompT: Strains harboring this mutation are deficient in outer membrane protease VII, ...methylate DNA. dcm: Similarly, strains with this mutation are unable to methylate cytosine within a particular...Tetracycline RecA-deficient; best for plasmids with repetative sequences.  F- ompT lon hsdSB (rB- mB-) gal ... of unstable proteins Tuner (DE3)   Contains mutated lac permease whch allows for linear control of ...Contains highly active thioredoxin reductase and glutathione reductase to faciliate proper folding; may increase...

...and CRISPR/Cas9 have also been used to create mutations in genes essential to HIV-1 replication. However...CRISPR therapies through PAM or seed sequence mutations. Multiple gRNAs could be used to combat this problem...LTRs or essential genes. Many of these escape mutations were located near the Cas9 cut site, leading Wang...escape occurs in an animal model. If these escape mutations are frequent, the dCas9-mediated “shock and kill... Zhang T, Li F, Yang W, Kaminski R, Fagan PR, Putatunda R, Young WB, Khalili K, Hu W. CRISPR/gRNA-directed... S, Wainberg MA, Liang C. CRISPR/Cas9-Derived Mutations Both Inhibit HIV-1 Replication and Accelerate ...

...CRISPR screens in immune cells have identified many putative regulators of immunity. However, in vitro screens...approaches; (4) encodes a Thy1.1 selectable marker to facilitate the isolation of modified cells; and (5) is compatible...molecular identifiers, which can improve the statistical power of in vivo screens. Figure 1: Using...used the pRDA_526 vector to knock out 30 genes putatively involved in CD8+ T cell responses to cancer. ...pRDA_526 vector. Moreover, the screen identified putative novel regulators of CD8+ T cell responses in tumors...

...that CRISPR's could be used to correct disease mutations, both in mice and in human cells. But that important...genes in one step without producing off-target mutations," Sha was quoted as saying when the news first...blogger Caroline LaManna put it well: "Wow, custom mutations in monkeys already. I know we've discussed it ...news was circulating. Scientists at North Carolina State, in the labs of Chase L. Beisel and Rodolphe Barrangou...structure may help us engineer around the current limitations of the Cas9 complex," said study author F. Ann...

...positive and negative controls to ensure proper interpretation of results. A positive control is generally...the precision and accuracy and therefore the statistical power of an experiment. Technical replicates ...autofluorescence, also interferes with data interpretation and can be addressed with a label control. ...autofluorescence and should be considered in the interpretation of results. Include the label control for all...membrane. A loading control is critical for data interpretation, especially when changes in protein expression...Waters, Jennifer C. “Accuracy and Precision in Quantitative Fluorescence Microscopy.” Journal of Cell Biology...

...Bicistronic vectors combine excitation and inhibition by using both excitatory and inhibitory channels activated...light within a specific range of wavelengths. Excitatory channels allow positively charged ions such as... the opsin.      Figure 2: Diagram of excitatory (ChR2) activation by light showing ions flowing...signaling cascade that may go awry in a disease state.      Figure 4: Example of an intracellular..., H. (2019). Light-activated chimeric GPCRs: Limitations and opportunities. Current Opinion in Structural...

...and a transposase. The piggyBac™ transposase facilitates the integration of the transposon specifically...completely seamless manner, leaving no sequences or mutations behind. Furthermore, piggyBac™ offers a large ...achieved using the piggyBac™ transposase, which facilitates direct integration of the donor-transposon (carrying...standard homology directed repair template to facilitate the selection of cells containing your desired...excision-only piggyBac™ transposase (PBx), which contains mutations making it competent for excision, but defective...primarily two other ways to create specific point mutations by gene editing. First, instead of using piggyBac...

...to be placed into the backbone in the proper orientation. For instance, if you were cloning a gene into...and HindIII. To place your gene in the proper orientation downstream of the promoter, you can add an EcoRI... promoter and placing the gene in the proper orientation. You then add ligase to the mixture to covalently... the backbone in either a forward or reverse orientation so you’ll need some way to verify that the insert...generally lyse the bacteria; add chemicals to precipitate out the high molecular weight genomic DNA; filter...your ligation, then you will need to verify the orientation of your insert. You may want to design a diagnostic...

...= transposon right-left orientation. T-LR= transposons left-right orientation. Image from Leo Vo.   ... its preferred target integration site: TNA(C/G)TTATAA(G/C)TNA. Tol2 can deliver 10-11 kB to mammalian...that detect loss-of-function or gain-of-function mutations. In these screens, the transposons encode reporter..., and have potential to overcome some of the limitations of viruses. TEs have a large payload, up to 100...such as the ~11 kB cDNA for dystrophin, the gene mutated in muscular dystrophy, require truncation to fit...

...for your protein has been mutated using CRISPR/Cas9. You check the mutation and it causes a large deletion...expression of your target or CRISPR/Cas9 to create mutations that essentially eliminate expression of the target...Capture MS Strategies This approach uses immunoprecipitation to capture proteins from a sample using the...analysis. Example: You are planning an RNA-immunoprecipitation experiment and want to validate that your... protein of interest. So you perform an immunoprecipitation with your antibody and send the captured ...

...pair, and flips the transgene into the correct orientation for transcription. Upon recombination one of ...no longer able to flip back into the inverse orientation (See Plasmids 101: FLEx Vectors). While this ...'s say the lox2272, that are in the opposite orientation (direction of arrows) the inverted transgene ...transgene is reverted into the sense orientation and one of the unused (loxp) sites will be flanked by two lox...lox sites now in the same orientation. Next, the loxp site is excised since it is between two lox2272 sites... sites in the same orientation preventing the transgene from reverting. Figure created with BioRender....

...with local representatives, or even run for office yourself! Plus, at the federal or state level some ... data gathering, regulatory efforts, policy implementation, program management, communications, diplomacy...branch or in Congress, or can also happen at the state and local level. The big secret about science policy...your career through engagement with your local or state governments. Most communities have opportunities...available science policy fellowships in federal, state, and local governments, professional societies, ...

...By mutating one of two Cas9 nuclease domains, researchers created the CRISPR nickase. Nickases create...SpCas9 nickases as examples for this post. The D10A mutation inactivates the RuvC domain, so this nickase cleaves... only the target strand. Conversely, the H840A mutation in the HNH domain creates a non-target strand-...accomplished with either a PAM-in or PAM-out orientation. Just as the names imply, PAM-out designs have...there are no available guides close to the desired mutation site. Quick tips for nickase design Ready to ...

...gene and check for any mutations. Once we identify the gene, we can easily annotate it by making a custom... “Detect Common Features” function to find and annotate promoters, selectable markers, tags, origins of...data in depth. You will see the same features annotated in our plasmid maps, including our most popular..., but you will also be able to create custom annotations and design additional experiments like restriction...

... to stably archived large-scale datasets by computational biologists. Figure 1: The BioGRID Open... in 629 cell lines, each manually curated and annotated from more than 45 publications, beginning with...and other details relevant to screen result interpretation. The analytical method for score assignment...   Screen results are reported as the original quantitative gene-level scores provided by authors rather...maintain consistency with published screen interpretations and allows users to query screen data without...

...frequency can indicate behavioral state, for example, if someone is awake, agitated, or asleep. Though DA firing...inserted into the region of the brain where DA quantitation is desired (Figure 1). The probe perfuses liquid...probe then collects the perfusion liquid for quantitation. The advantage of this method is that it can...performed in a live awake animal by surgical implantation of the probe into a specific brain region. While...

...develop these fibers, the labs looked at previous implementation of optogenetic tools, calcium sensors, and ... be, as well as other features necessary for implantation in brain tissue. The gut, on the other hand...fibers could not be directly repurposed for gut implantation. The fibers they ended up generating were 10...digestion of food, while brain tissue is relatively static). To operate both types of implanted fibers simultaneously...neuron activity for at least several months after implantation of fibers, indicating the system was indeed ...

...containing the desired mutation but lacking both RUBY and the construct that rescues the mutation. I will be able...identified by a phenotype caused by the segregating mutation. Selecting these plants is a learned skill that...do this by expressing a gene that rescues the mutations, using RUBY as a selection marker. These plants...because they will no longer show the effects of the mutation. But ¼ of the offspring from these plants will...

... and implantation of the optical fiber into the region of interest 3) Behavioral experimentation I’ll ...not to say that the technique doesn’t have its limitations or that other technologies need to be discarded...what are the methodological and experimental limitations and considerations? I recently spent two months...Intramural Research Program (NIH-NIDA IRP), with the stated goal of learning behavioral optogenetics in order...viral injection and commencement of behavioral experimentation.  Whether you attempt to stimulate the cell...

.... phaffii Growth properties Facultative anaerobe Facultative anaerobe Obligate aerobe Number...applications in food science and biotech due to its fermentation abilities and other useful traits. The “biotech...the preferred pathway for generating knock-in mutations, and NHEJ is an error-prone pathway frequently...frequently introducing frameshift knockout mutations. Thus, if genetic manipulation one way or the other (knock-in...

...endeavor, a map of possible TF-induced cellular states was generated (Joung et al., 2023). Fig....generated by the human fetal expression atlas. Computational analysis was used to infer differentiation trajectories...example, very few diseases manifest as a single mutation or expression dysregulation event. Diseases are...multiple layers of dysregulation from either genetic mutation and/or TF dysregulation. A targeted TF screening...differentiation), and 15/17 morphologically aligned. Documentation of the network that initiates biological reprogramming...

...library amplification and sequencing. Maintain representation of the library during transformation First,...sequencing Once the DNA has been extracted, the representation of the library should be confirmed using next-generation...counts. You can use these counts to confirm the representation of your library. When plotted as a histogram...uniformity (see Figure 3A). Addgene finds that this representation is most easily understood visually. As an example...careful amplification and maintenance of good representation. Addgene wishes you luck in your future library...

... that these mutations clustered in a particular region of the R-body. Further mutatgenesis of this region...understanding these complex processes can’t be overstated, we can also learn a lot from Nature’s simpler...dynamically rearranging into different polymerization states. Instead, R body monomers respond to pH changes...

...plates that don’t respond as expected, use of representative images, measuring the main outcomes in several...practices (QRPs) can quickly lead us to find statistically significant results that are not robust. Sampling...each hypothesis will be tested with a specific statistical test.  Preregistration makes clear the distinction...to present that work using hypothesis testing statistical tools that generate a p-value.  As an example... of Life Sciences at Rutgers University. His dissertation and research background is in citizen science...

...vivo and in vitro, as well as to insert targeted mutations through homologous recombination. To further increase...protoplasts, Xie et. al observed slightly higher mutation rates than they’d previously reported in protoplasts..., they found that 50% of plants had biallelic mutations at all five loci. This highly efficient multiplexing... space of a standard pol III-gRNA, which will facilitate CRISPR/Cas9 multiplexing in systems with limited...overlapping activity. Multiple gRNAs can also facilitate deletions of other regions, such as noncoding...

...trick is to precipitate the RNA using lithium chloride. LiCl solutions selectively precipitate RNA, but ...cesium-chloride gradients, water-saturated phenol, sodium acetate, and chloroform are added to the homogenate and...is extracted and RNA can then be isopropanol precipitated. This method reduced the time it took to isolate... results of a downstream application such as quantitative PCR to assess gene expression. There are several...

...load The Wikipedia definition of cognitive load states: “cognitive load refers to the used amount of working...re unaware that we are leaning on an unspoken expectation of our audience knowing foundational concepts...feedback in a variety of ways, like having them restate or summarize the concepts you’re covering. Don'...just really tired and needed a slower, simpler presentation than I had planned; other times students were...fired up and enthusiastic and needed a faster presentation with more information than normal. I had to ...

...DNA-protein interactions or to analyze chromatin states (CHIP-seq, FAIRE-seq, Cut & Run) and to detect ...interactions has lagged behind, likely due to limitations of current means to detect RNA-protein interactions...interacts with RNA. Methods like cross-linking immunoprecipitation (CLIP) identify RNA-protein interactions ...such as during drug action or cell signaling. Limitations of RaPID Like other BirA*-based proximity labeling...early-onset cataracts, is characterized by point mutations in the Iron Response Element (IRE) RNA motif of...