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...Varmus H. Retroviruses. Cold Spring Harbor Laboratory Press; Cold Spring Harbor, NY, USA: 1997. PubMed...them attractive and tractable tools for genome engineering. Scientists have engineered viruses to target...incompetent viral particles, are required. We will be covering these safety measures in a separate, upcoming ...breakdown of the nuclear envelope, which occurs during mitosis. The most well-known retrovirus is HIV....PubMed PMID: 21433340. 4. Cronin J, et al. “Altering the tropism of lentiviral vectors through pseudotyping...

...educational content Addgene truly believes that sharing speeds science, and our entire mission is to enable...informative content to help researchers in creating and sharing knowledge, related to the types of materials we...help.addgene.org, where you can easily search articles answering all of our frequently asked questions!    ...published alongside our new antibody services in the spring of 2022. The first step was to understand information...licensed and cited figures from papers or image sharing repositories. Then we format, do a final review... and knowledge changes and require updating or retiring. CRISPR/Cas9, for example, has gone through rapid...

...to keep their cargo below 4.4kB (with the ITRs bringing total genome length to ~4.7kB.  If your insert... kB, then you would be oversized even before considering the need for some manner of polyA. In this scenario...intein approach.   Q3: What’s the best method for titering AAV? Is there a difference in titer results between...equal animal dosing, we advise that users keep titering method consistent across samples within a project...microglia in vivo.   Q5: What is the efficiency of co-delivering different AAV particles into the same cells in...any difference in transfection efficiency when delivering these viruses via retro-orbital vs. tail vein...

...and Keira Havens, co-founders of Revolution Bioengineering. Read their previous blog post about how they...directed at GM crops has burdened this emerging engineering technology. Monoculture and chemical spraying... you have developed a genetic tool for plant engineering, please share it with Addgene to both build the...biology community and to support more efficient engineering of a better tomorrow. Thank you to our guest...outside of Washington D.C. and has always enjoyed exploring the outdoors and experiencing nature first-hand...SynBio Reading: Learn more about Revolution Bioengineering Follow RevBio on Twitter Like RevBio on Facebook...

..., too. And remember, if you discover during grad school or during your postdoc that you enjoy and have...engineers were attending the same meetings and hearing the same words, they were leaving these meetings...know what kind of plate they were talking about during the meetings. And “plate” certainly wasn’t the ...experimental protocols. Clarify: Asking for clarification during a meeting can help get everyone on the same page...have come upon any other effective approaches for clearing up communications lines in your professional life...

...Challenges of engineering channelrhodopsins There are three main challenges to engineering channelrhodopsins...channelrhodopsins with high-throughput proteins engineering methods, like directed evolution, because channelrhodopsins...-stimulate” their VTA dopaminergic neurons by triggering a burst of light that could activate ChRger2 ... require fiber-optic cannula implantation, but altering ChRgers so they can be activated with tissue-penetrating...2019) Machine learning-guided channelrhodopsin engineering enables minimally invasive optogenetics. Nat ...

...or little deterioration or change of the signal during the course of the experiment. The best fluorescent...photobleaching rates (Shaner et al, 2008). This brings us to the key question: what is the best way to...photostability of different fluorescent proteins? Measuring photostability Since it is clear that a photostable...measurements for different fluorescent proteins and by comparing how the fluorescence intensity changes over time...than predicted by these high power experiments. Measuring photostability at realistic powers will provide... but they do not mimic the natural situation. Measuring photostability in living cells provides a much...

...replication of virus and its infectivity in check. Engineering controls, safety systems that are built into ... you can receive appropriate treatment and/or monitoring. Virus-specific concerns   In nature, some viruses...protection! When centrifuging viruses (a necessary step during production), there is a significant risk for aerosol...of following your lab’s biohazard policies. Administering Virus Many virus experiments involve needles...accidents is not following safety protocols.) If administering virus to mice, part of your preparation should...let you work with these valuable tools without endangering yourself or others. Happy transducing! Resources...

...Here's what you'll find in this post: Epigenetic engineering with single-pot assembly Tracking cell-cell interactions...New items from the viral service Epigenetic engineering with single-pot assembly By Eric Perkins Though...epigenetic researchers custom design their own engineering plasmid tools in about a week. Taking advantage...receiver cells. What’s more, G-baToN can be used to bring cargo, such as protein or DNA, to manipulate function...CRISPR Plasmids and Resources page.  Plasmids for delivering CRISPRi components by a single AAV vector are...Choudhary's lab developed a chemogenetic system that brings Cas9 to ubiquitin ligase for degradation.    ...

...There are a few different issues that can arise during the plasmid purification process. One is the possibility...broken into a few broad stages: lyse the bacteria harboring your plasmid; separate plasmid DNA from unwanted...environmental impact, check out Addgene’s blog post on bringing sustainable practices for the lab. Plastic consumables...from bottles and the overall weight of the package during shipping. Of the kits reviewed here, NEB’s Monarch..., so be sure to check with your lab manager or ordering department for these.  While budget is an important...Different Uses Plasmids 101: Optimizing Plasmid Yields Bringing Sustainable Practices to the Lab: Easy Action ...resources on addgene.org DNA Quantification Protocol Recovering Plasmid DNA from Bacterial Culture Protocol ...

... the abdomen. They are a popular choice for administering different drugs and viral vectors. Like IV injections...suggests, in the muscle. Common uses include administering vaccines, pain meds, or other drugs. Identifying...don’t always require needles. If needles make you cringe or your experiment will allow you to avoid poking...straight to the stomach. This technique involves administering medication through a feeding tube with a bulb...introduction to common injection routes in mice! When considering what method you’d like to use, remember that ...Organisms Additional resources on Addgene.org Genome Engineering Guide CRISPR Mammalian Expression plasmids Cre-Lox...

...tissue clearing Similar to light microscopy, there have been significant advances in tissue clearing techniques...brightness with increasing depth caused by light scattering and absorption.  Finally, the photomultipliers...techniques during this century.  The first successful attempt to render fixed anatomical preparations transparent...Ultramicroscopy with tetrahydrofuran-based tissue clearing (13,14). A few years later, Karl Deisseroth’s ...  The combination of LSFM with multiple tissue clearing strategies and neuronal labeling methods will ...

... parents in STEM careers, chances are they’re wondering how they could contribute to the school, too. ...Middle school: supplementing the science curriculum Sharing my expertise with our district middle school was... an authority in this area. What is genome engineering? How does CRISPR work? This section was essentially... of the talk by running it by the teachers and sharing some slides on Twitter, where I was able to get...computers and the internet before it, genome engineering technology is moving along much faster than it...understanding genetics is so timely.” Genome engineering, and many other technologies and fields in biology...

...the non-radiative transfer for energy between neighboring fluorophores, FRET can be used to detect the ...ratios) to generate useable data. What’s more, measuring and quantifying FRET is often easier said than...complete FP because the PIPs they’re tethered to bring them into close proximity (Figure 1). Formation ...independent of PPIs. Overexpression can be assessed by comparing the expression levels between BiFC constructs ...Analysis of Protein Interactions in Live Cells. Cold Spring Harb Protoc 2013:pdb.prot076497. https://doi.org...Fluorescent Protein 101 blog posts Read our blog post covering an introduction to FRET Resources on Addgene....

...by loss of protein during digestion, or masking of epitopes during sample anchoring and gel polymerization... Tillberg, et al. 2016). That being said, the anchoring, digestion, and expansion steps can impact the...occurs, due to a reduction in target concentration during expansion. One group has found that using Fab fragment...et al (2023) Magnify is a universal molecular anchoring strategy for expansion microscopy. Nat Biotechnol...

...where droplet digital PCR (ddPCR) comes in. AAV titering using quantitative PCR Before we dive into the... PCR is complete, the results are analyzed by comparing the threshold fluorescence of the viral DNA to...reasons, we are in the process of transitioning our titering method from qPCR to ddPCR. Droplet digital PCR...reference, meaning saving on reagents (and time)! AAV titering using droplet digital PCR Droplet digital PCR ...generator, you will have to be careful about transferring your droplets from the droplet generator to ...detection of microbes. Some applications include measuring viable probiotics and detecting circulating pathogens...

...held. Imagine this situation: You’ve spent hours scouring a company’s LinkedIn pages, given yourself the...guaranteed to get the job, however, many times the hiring team will also want to examine not just technical...technical skills, but also transferable skills, before offering someone their dream job; They want to know if ... and to demonstrate them to the people who make hiring decisions. Though having all the technical skills...get a great snapshot of the skills that you can bring into pretty much any other workplace. To get your... for non-academic science careers Learn about mentoring Resources on Addgene.org Check out our careers...

...Knight TF. Engineering BioBrick vectors from BioBrick parts. Journal of biological engineering. 2008;2:5... fluorescent protein were fused in-frame to a tethering partner, which directed localization of the fluorophore...Construct Fluorophore Excitation/emission maxima Tethering partner Subcellular localization 1 TagBFP 399nm...Constraints on the order of the modules, because neighboring fragments require compatible cohesive ends Engler...biological part assembly. Journal of biological engineering. 2010;4(1):1. Pubmed PMID: 20205762. PubMed Central...

...are syringe or vacuum infiltration. Syringe infiltration (D'Aoust et al., 2009): Usually a syringe without...contains a plasmid with a T-DNA (transfer-DNA) region. During infection this T-DNA region is transferred into...transformation and gene expression can be tracked. During a transient leaf transformation, the engineered...Transient expression of antibodies in plants using syringe agroinfiltration." Recombinant proteins from plants...

... You need to balance respectfully answering questions while steering the conversation back to your vision...proposal or a 10-year research plan. I suggest preparing for both types. Your research vision is your approach...proposal will distinguish between competing models. Comparing model A vs. B is more constructive than testing...vision. On the day of your chalk talk Bring your own fresh set of erasable markers. You’ll have the colors...and consider eating one before your chalk talk. Bring your own bottle of water. If possible, write down...