...safe for clinical applications. Excisable Cre-lox lentiviral vectors: This approach is the same as using...of excision, this approach is still not considered safe for therapeutic applications. piggyBac (PB) transposon...This is considered a safer approach for developing therapeutic applications, but it can take a long time...time, scientists have used almost all available approaches for generating iPSCs. The generation of iPSCs...Retroviruses are not considered safe for clinical applications.     Lentiviral: Lentiviral vectors infect dividing...dilution via cell division. A drawback to this approach is it has lower levels of efficiency at generating...express reprogramming factors for therapeutic applications. Sendai viral vectors: Sendai virus is a single...

...relationships. However, most job seekers will and should apply for jobs posted online as one part of their job ...Utilizing existing relationships as part of the job application process can dramatically increase the chances... of a resume can be crucial for a successful application. It takes time to prepare an excellent resume... Many job postings will result in hundreds of applicants the day they are posted.  If your resume is not... here for more on timely submission of a job application. 3) Read job descriptions carefully Be open minded...have to have all of the listed qualifications to apply for a position.  A job description is usually a ...wish list and the hiring manager knows that no applicant is likely to meet all the criteria. Note the criteria...

...skipping approach in mice, DMD researchers are very excited. This approach may also be applicable to a number...success in basic research, researchers are eager to apply it in a clinical setting. CRISPR is often used for...), an X-linked recessive disorder affecting approximately 1 in 5000 males. DMD is caused primarily by ...very large (79 exons), but much of the sequence appears to be nonessential. Within the mutational hotspot...so researchers have set their sights on another approach. Since shorter forms of dystrophin can still be...researchers have begun to explore genome editing approaches for exon skipping. Addgene depositor Charles ... removal rate of exon 51, which resulted in appropriately localized dystrophin. In a subsequent study,...

...genes provided by the user. This approach is useful for several applications as will be discussed below. The... screen approach is to use smaller libraries targeting subsets of genes. With such an approach, you lose...custom CRISPR screen and the appropriate phenotypic readout, similar mapping could be rapidly performed ...challenges with a custom screen approach is to identify an appropriate list of genes to target. Thus, ... involved in a studied cellular behavior. This approach is particularly suitable for in vivo screens, ...drugs. Known drug targets  Another interesting approach is to design screens against known druggable targets...preferentially those for which validated inhibitors or even approved drugs already exist. If you are lucky and find...

... tools for familiar applications. But how does one invent a tool for applications that don’t even exist...and develop potential applications.  Andrew is a frequent collaborator and is happy to give advice and support...phenomenon, improve the tool, and develop new applications for magnetoresponsive proteins. Motivated by...organisms) are opaque, which makes most biomedical applications of these tools difficult or impossible. But ...tool with magnets would enable new research and applications that don’t yet exist, but it wasn’t clear that...molecules like proteins with magnetic fields generally appears to be weak — if they exist at all. Andrew and Maria...fluorescence intensity decreased whenever the magnet approached the sample (by less than 1%) and recovered after...

...single model. The first approach can lead to new directions, while the second approach could terminate with... Many thanks to our guest blogger, Erik Snapp! Erik Lee Snapp received his PhD in Molecular Microbiology...This post was contributed by Erik Snapp, the Director of Student and Postdoctoral Programs at the Janelia.... Given this large sum, one might expect that applicants would work for months to hone a chalk talk. Yet...preparing for both types. Your research vision is your approach to resolving a significant knowledge gap in your...has unclear utility. You cannot predict what will happen. Without the prediction, you have no hook. A battery... you need to be able to describe experimental approaches in terms of the question, the methods, the tools...

...our guest bloggers Erik L. Snapp and Lindsey M. Costantini! Erik Lee Snapp received his PhD in Molecular... post was contributed by guest bloggers Erik L. Snapp and Lindsey M. Costantini. "You underestimate the... of a mystical "Force," this quote is equally applicable to many microscopy experiments with fluorescent...not fluoresce. A dark population is not readily apparent and can confound quantitative imaging experiments...for use in cytoplasmic environments. However, approximately 40% of human (and most eukaryotic) proteins ...have reported that FP cysteine residues form inappropriate disulfide bonds in the secretory pathway (Figure...localization. For example, the formation of inappropriate inter-chain disulfide bonds between FPs blocks...

...developed and applied. The current pandemic serves as a striking example of the applicability and importance...research. I applied, hoping that the experience would improve my scientific and application writing skills...science (as opposed to translational, biomedical, or applied sciences), people conduct research to answer questions...revelation. Science in any field can generally be applied to improve our lives and solve global problems....grants Varying degrees of engagement with what happens in the lab Official title is typically Professor...the people around you. Be proactive Rather than applying only to posted job offerings, the best research...ties into the previous one too. I would recommend applying for multiple positions—don’t put all your eggs...

...received 116,224 employment based green card applications and approved 91,023 during the first 3 quarters of ...options discussed in this post benefit from the applicant thinking about the process early, keeping good...is a numerical quota of 85,000 total H-1B visas approved each fiscal year: 65,000 visas plus 20,000 reserved...occupation" as requiring theoretical and practical application of a body of highly specialized knowledge in ... April 1 deadline for companies to submit an application on their behalf.  If an individual obtains a ... cap subject H-1B (last year less than 30% of applicants received sponsorship) then he or she can start...1B petitioners are exempt from the cap if the applicant works at institutions of higher education, a related...

...real-time PCR (4). Restriction mapping and modern sequencing Restriction mapping informs on DNA sequence through...useful applications and have informed many current techniques. For example, restriction mapping led to...historical techniques: southern blotting, restriction mapping, and sequencing gels, as well as their modern equivalents...comparison to a control sample. Southern blotting applications range from confirming the results of cloning...gene that causes sickle cell anemia. The mutation happens to destroy a sequence recognized by the restriction... was commonplace, researchers used restriction mapping to investigate DNA sequence content by comparing...of the DNA. Starting in the 1970s, restriction mapping was commonly used to map DNA and examine genomes...

...zebrafish embryo has been mapped using single cell RNA sequencing approaches (Harland, 2018). Several ...investigations. Genetic resources for zebrafish A common approach to unravel the gene networks that drive biology...DNA into the genome is useful for a variety of applications. For example, specific tissues such as blood... regions is also possible using an alternative approach that exploits the bacteriophage integrase phiC31...useful, I’ll outline two recent lineage tracing approaches that led to important insights in developmental...tracked using microscopy and advanced computational approaches (Keller et al., 2008). This microscopy-based ...pathways control this process. Additional zebrafish applications and conclusions Zebrafish are also popular outside...

...subsequent, repeated activation), this is an approximation of reward or the sense of pleasure that the ...EthoVision. Consult the literature to find all the applications of optogenetics relevant to your field. Ultimately...behavior, certain technical considerations still apply. Testing stimulation First, In lieu of electrophysiology...stimulate them for 30min to an hour and observe what happens. You never know how stimulation of a particular...optimization, turn on the laser and watch what happens. Who knows, you may observe something that could... discovery. Monitoring behavioral output The appropriate behavioral readout for your optogenetics experiment...brain region that may be important in memory, an appropriate behavioral test might be contextual fear conditioning...

... a bioinformatics approach examining genomes possessing CRISPR systems with apparent self-targeting. Two... plasmid or group of plasmids' functions and applications. We hope that these articles make it easier ...includes four promoters, six fluorophores with nonoverlapping emission spectra (N- and C-terminal tags for...Targeting and manipulating interneurons using a viral approach Genetically modified organisms like transgenic...successfully targeted via viral particles, an effective approach to target specific neuronal subtypes was not available..., Gordon Fishell’s lab has developed a new AAV approach using regulatory elements that restrict expression...tools in virtually any vertebrate. This novel AAV approach allows you to target and functionally manipulate...

... matches to your insert. Select the appropriate gene if it appears in the Entrez Gene list, as this will...would search for it in PubMed and choose the appropriate publication. This publication will be listed ...preprint. In the case of a preprint submission, we are happy to update the publication information once the manuscript...pass our standard QC process and the MTA has been approved. The spreadsheet option is recommended for ...contain epitope tags or fusion proteins that are appended to new inserts through cloning. *Note — If you...complete these pages sequentially by entering the appropriate data and pressing "Save and Continue to Next ...around to the different pages by clicking on the appropriate boxes at the top of the page. Pro Tip! Be sure...

...Cas9 in mammalian cells, many of these principles apply to using CRISPR in other organisms. First, consider...a good way to narrow down which reagents are appropriate for a given experiment. Make sure to check whether...may not be a perfect plasmid for your specific application, and in such cases, it may be necessary to customize...reagent to suit your needs. Genetic Manipulation Application Cas9 gRNA Additional Considerations Knockout ...Cas enzymes increase specificity. Dual-nickase approach increases specificity but is less efficient. Each...viral delivery of CRISPR reagents may be more appropriate. In cases where off-target editing is a major...method Expression System Components of System Application Mammalian expression vector Cas enzyme promoter...

...the filament from (a-b), showing the appearance and disappearance of 649-mDia1 or 549-CP (upper, middle...block its catalytic activity and instead become trapped in a covalent bond with its substrate. Then, all...having to clone a whole new construct for every application. Plus, the photophysical properties of chemical...advantages over fluorescent proteins, opening up new applications.   Figure 1: Live-cell labeling strategies...acids 1 Various Many Complicated   Applications Organic dyes are typically both brighter and...self-labeling tag and a fluorescent protein) enables applications like multi-color imaging (Figure 2). If you’...Actin, cyan), with formin (649-mDia1, magenta) and capping protein (549-CP, yellow) at the tip. 649-mDia1 ...

...depending on the conference you’re attending and who approaches you. You might also have cross-team projects ...communication. Although these real world examples did not happen in the lab, they have parallels to collaborative...types or purposes of each given container, when applicable Any other relevant information Prescribing an...misinterpretation, that misinterpretation was definitely happening. Our solution for this problem was the creation...lab setting, where student and postdoc turnover happens regularly and it behooves everyone in the lab to...potentially ambiguous terms. Defining words and applying annotation can be particularly useful in academic.... But if you implement it well, here’s what can happen: More effective meetings. When everyone at a meeting...

...truth is, there is a lot of software development happening at Addgene under the hood. A significant portion...this can lead to wildly different ideas of how to approach a problem, but it is these very differences that...similar cognitive biases and this would become apparent in the software we develop. We would all have ...framework is based on an iterative and collaborative approach to development. Scrum teams plan and break up ...are also able to consider different technical approaches that may come up during discussion.  One example...perspective which in turn helps us decide how to best approach it in our code.  Sometimes while thinking through...that the clarifications lead to the story being scrapped or broken up into smaller stories. Since this ...

...virus If having happy cells is the first step of a successful lentiviral infection, happy virus is the second... express a gene of interest. While the general approach for using lentivirus, infect and select, seems... cell lines, ELISA, fluorometry, or PCR-based approaches to detect even low levels of contamination. For...Uphoff et. al. outline a do it yourself PCR-based approach complete with freely available positive controls...old. Ensuring that you are working with healthy, happy cells is one of the easiest ways to improve both...envelope. One drawback of the multiple harvest approach is that it tends to result in a higher volume ... freshly collected lentivirus for downstream applications. While this ensures that the highest titer virus...

...non-refundable fee is applied to cover processing costs. Once the application is approved, Addgene then ships...excited to announce we now offer an export license application for rabies materials! Some of the materials in...international requestors. We also offer export license applications for VSV and SARS-COV-1 plasmids. Export control...license process    An export control license, once approved, allows Addgene to ship plasmids or viral vectors...will need to provide a brief description of the applicable research project when purchasing export-controlled...export-controlled materials. Addgene will then apply for the license for distribution to the requester’s organization...offered VSV and SARS-COV-1 plasmids under this application process since May 2020. We find this process ...