...tissue-specific diseases, as you can target your library to a significant degree using different types of...       In vivo, in vitro, ex vivo CRISPR screens can be carried out using a few different methods — in...infected with the CRISPR library. In vivo screens can become costly, so having an efficient and effective...delivery method for in vivo CRISPR screens. AAV vectors can be made at high titers equal to those achieved with...affect which is chosen for certain experiments. AAV can be safely handled at BSL-1, while lentivirus requires...vectors being packaged using VSV-G. In contrast, AAV can be packaged in a variety of capsids. To date, at ... interest — for example, AAV8 for kidney cells — can help improve infection rates in your experiment. ...

...secondaries, can improve signal strength (Sherry and Stiles, 2022). Fourth, signal continuity can appear reduced...they can achieve. This means that regardless of how powerful of an objective you have, you can’t resolve...is limited in the number of specific targets you can label in a sample. Super-resolution microscopy (SRM...original size. Depending on the protocol, samples can be expanded to ~4-10x linearly! This expansion physically...said, the anchoring, digestion, and expansion steps can impact the integrity of different targets and labels..., et al. 2020). With any variation, your samples can tear or expand unevenly, introducing distortions ...expansion results in dimmer samples. Reduced signal can be caused by loss of protein during digestion, or...

...fluorophore can emit a limited number of photons before bleaching. The amount of information one can extract...overexpression of protein fusions to gene-edited cells can decrease the number of fluorophores, compromising...budget, which determines how long and precise we can track individual molecules. Poor photon budgets are...fluorescent proteins—even the thriftiest cell biologist can feel like a college student searching couch cushions...more photons from a sample. Chemical fluorophores can be substantially brighter and more photostable than...transcyclooctene–tetrazine)– Nonnatural amino acids can be incorporated into a protein structure and then...1e) are inherently fluorogenic and several others can be made fluorogenic using environmentally sensitive...

... of a particular observation can be easily studied as each component can be examined independently. In...thus modeling genome replication. These products can be detected by Northern blot analysis. In addition...producing firefly luciferase. Firefly luciferase can readily be measured to quantify (indirectly) transcription...by the viral polymerase. Alternatively, the mRNA can be detected directly by Northern blot analysis or...In addition, experiments can be performed in the absence of some viral proteins (compared to viral infection...Therefore, the roles/functions of individual proteins can be studied independently of other viral proteins ...simplifying the interpretation of results. This system can be used to develop an understanding of and manipulate...

...the ‘bait’) can be ‘fished out’ for further study. In Y2H screens, a group of prey fusions can either be...experiments. False positives can occur even in small scale experiments, although these can often be accounted for...cellular compartment or growth stage). A prey library can either be applied to the bait in a single population...false negatives; up to 75% of known interactions can be missed by single-pass two-hybrid screening (6)...levels of bait and prey proteins - overexpression can lead to false interactions. Lowered expression levels...for problems with one or more of these parameters can lead to a high number of false negatives in Y2H screens...responsible for the modification in the assay host strain can help. In order to reduce the chance that the configuration...

...titers can drop 10-fold for every 2kb increase in insert size. To overcome this, lentivirus can be concentrated...popular gene delivery tools, producing lentivirus, can pose certain challenges. Whether choosing a system...competent lentivirus (RCL). Replication competence can arise from recombination events during viral production...fourth plasmid. The requirement for additional DNA can impact the transfection efficiency of the packaging...Requiring an additional plasmid for transfection can make these optimization experiments a bit more challenging...transfection conditions. As mentioned above, plasmid ratios can be adjusted to enhance titer. In addition the transfection...for an example of how changing transfection ratios can affect transfection efficiency. For some transgenes...

... primary cells, embryos, and animals. Nanoblades can also carry different types of CRISPR cargo including...Nanoblades then bud into the cell culture supernatant and can be purified by centrifugation. Gag-Cas9 fusion protein...the two proteins, thus releasing Cas9. Nanoblades can also deliver Cas9 variants, such as a transcriptional...Gag-Cas9 fusion protein. A single Nanoblade particle can be loaded with 1-4 different gRNAs. Viral envelope...showing that the method of Cas9 and gRNA delivery can influence the level of off-target effects, with electroporation...carries a gRNA, Cas9, and a HDR donor template DNA can be generated by incubating Nanoblade particles with...zygote which is both technically challenging and can negatively impact zygote viability. Nanoblades, however...

... where one can publish brief novel findings. These results are normally one figure and can be negative...Using SapTrap, this CRISPR plasmid can be created in a single tube. You can also find additional SapTrap plasmids...and Sibernetic, a fluid mechanics simulator that can be used to simulate muscle tissue as it applies to...microPublications are peer-reviewed and assigned a DOI that can be referenced. In addition the data published in ..., 2015). In this system a modified TIR-1 protein can degrade degron-tagged proteins in an auxin dependent..., a GAL-4 based bipartite expression system that can regulate gene expression in C.elegans. In this system...promoter controls the expression of a GAL-4 driver that can bind to activation sequences upstream of genes to...

...remodeling can occur by two main mechanisms, either through histone modifications, which can influence ...hunger that can lead to excessive eating and obesity. What is fascinating is that both syndromes can present...on top of the genome’). Epigenetic modifications can result in phenotypic changes without any change in... have very different structure and function, and can have a specialized role within an organism. Mechanisms...wrapped around histones changes, then gene expression can change as well. The more frequently and tightly bound...in the promoter region of tumor suppressor genes can repress their transcription and inactivate them. ...bisulfite sequencing. However none of these techniques can be used for in-situ research of dynamic modifications...

... available. Without a comprehensive resource, it can be overwhelming to choose a Cas for your experiment...planning, you need more nitty-gritty details — how can I multiplex this enzyme? What other RNA components...embedded in the References lists on every page you can find direct links to plasmids from the article if... a Cas entry (SpyCas9a) from CasPEDIA.   How can I use it? CasPEDIA operates like many search engines...For the advanced search options, this is where you can really identify the best Cas for the job! There are... are a series of drop-down menus in which you can manually enter details to refine your search (trans ...ability, gRNA design, etc.). With this feature, you can compare different Cas enzymes to make sure you are...

... kb, ample room for most experimental needs, and can transduce (infect) a wide range of cell types with... coming in around 9 kb. Unlike gamma, lentivirus can infect both dividing and non-dividing cells. Lenti...envelope or a post-transcriptional regulatory element can be used for either type of retrovirus. Adeno-associated... is a small, single-stranded DNA parvovirus. AAV can infect humans and some other mammals but is not currently...an extrachromosomal part of the host genome that can replicate and divide along with the endogenous DNA...high immune response as retroviruses. This virus can also transduce dividing and non-dividing cells, making...routinely being developed) to choose from, which can target the virus to specific cell types of interest...

... scientific and medical tools. As scientists, we can manipulate what this cargo is and where it goes, ...cells they infect. In the lab, this means viruses can be engineered to be cell type specific. Once inside...genome, however, isn’t enough to ensure the virus can replicate and spread. Instead, viruses replicate ...one they have a high transduction efficiency and can effectively infect a wide range of cell types that...difficult to transfect’, such as primary cells. They can also be directly injected into hard-to-reach organs...Lentiviruses are a genus of the retroviral family that can infect both non-dividing and dividing cells. They... this through nuclear localization signals which can traffic their viral particles inside the nucleus,...

...platforms such as Github, users can download the software for use, or they can modify and optimize the code...have been convinced that most microscopy techniques can be replicated with commercially available consumer-grade...feasibility of manufacturing, and subsequent assembly can be difficult to overcome. Universities may not have...representations of the 3D models, which in many cases can be produced quickly with the printer, act here as... since a part developed in Germany, for example, can also be printed quickly in Madagascar. Platforms ...across laboratory and disciplinary boundaries and can allow for innovative organizations and companies ...example of how sharing of data and molecular tools can lead to faster and more efficient science. Researchers...

...LentiCas9-blast virus can be used to generate Cas9-expressing cell lines, which can then be used both for...collection of tools so that researchers around the world can accelerate their work. After all, as we like to sayat...is the headliner of most CRISPR applications and can be expressed from your cell line of choice after ...nuclease, which introduces double stranded DNA breaks, can be expressed from lentiCas9-Blast. Purpose of LentiCas9...for generating your own lentivirus. Secondly, you can use a LentiCas9-Blast viral prep to create a single...University of North Carolina, Chapel Hill. These can be used to investigate the effects of activating ...Purpose of this AAV pAAV-hSyn-DIO-hM4D(Gi)-mCherry can be used to induce neuronal silencing in vitro and...

...which can be done through the align tab of our sequence analysis web-tool. Alternatively, you can use DNA-editing...available and provide some advice on the steps you can take to verify your own plasmids. Staying organized...This will ensure that future members of your lab can access and use the materials they need, and avoid...we have a standard backbone with one insert, and can use common primers to sequence from the backbone ... primers to sequence the important features. You can use primer design software such as Primer3 to help...first 50-100 base pairs of your sequencing read. You can expect to recover around 750 bp of clean sequence...analysis of a large number of sequencing reactions, you can make your life much easier by selecting the right...

...tags can be used to facilitate the visualization of specific proteins in cells. Tagged protein can easily...specific antibody and the proper microscope setup we can observe the whole population of one particular protein...proteins instead of methionine. The labeled proteins can be detected using click-chemistry that covalently...alkyne tag. The newly synthesized and tagged proteins can then be observed with fluorescence microscopy. This...Professor Schuman’s lab showed that FUNCAT’s usefulness can be extended to determine the turnover of a specific...of interest are in close proximity to each other can the DNA strands take part in rolling circle DNA amplification...discover new restaurants, go to concerts and bake. you can contact her at [email protected]. References ...

...secondary antibodies can be used with different fluorescent labels and both targets can be successfully visualized...region may be changed.   Chimeric antibodies can help A chimeric antibody is made by changing an antibody... 2: Two primary antibodies with the same isotype can not be used for multiplexed indirect staining methods... visualized (bottom panel).    Can any antibody be converted? Isotype conversion is a unique characteristic...sequences are known and encoded on a plasmid, they can be easily manipulated into more useful tools. In ...the binding specificity to the original target and can be used with secondary antibodies against the new... some sequence from their parental species which can be bound by broadly reactive secondary antibodies...

...piece of DNA so that it can be easily cloned into a plasmid of interest. You can use similar processes ...reverse-complement of this sequence so that we can successfully amplify the ORF. You can generate the reverse-complement...It is helpful to choose restriction enzymes that can both function in the same buffer, as this will save...restriction site to improve cutting efficiency. You can generally add any 6 bases, but you should ensure ... reverse complement to get PCR amplification. We can start similarly, taking the final 18 bases of the...there is very little chance for mis-priming, so you can use a pretty wide range of annealing temperatures...

...timely manner (within a few days or a week) so they can easily recall the encounter. As a side note, my guess...for awhile or ever so your LinkedIn profile link can be your new card. Follow your contact’s company on... about their new publications via Pubmed, so you can congratulate them when there’s good news.  Send an...an email if you have something to ask or say. It can be as simple as “it was nice meeting you.” Include...non-work topics and interests. Offer help when you can, instead of just asking for favors.  Don’t focus ...’s a waste of our time.  Networking connections can last years This is a graphic snapshot of my LinkedIn... network give rise to a starfish shape. You can ask me about the different arms of this starfish ...

...feature in reporter plasmids.  Learn How Luciferase Can Be Used In Concert with Fluorecent Proteins in Nano...promoters or enhancer regions on gene expression can be determined by detection of the reporter in a specific...scintillation counter, and the activity measurement can then be used to quantify the effect of the regulatory...luciferase or the use of alternative substrate reagents can permit measurements of luminescence from live cells...Monitor miRNA knockdown Expression of luciferase can be monitored as a downstream measurement of miRNA... luciferase activity. This experimental protocol can readily screen miRNAs for effectiveness. Analyze ...Signaling Pathways Known cellular signaling pathways can be investigated in a high-throughput screen using...