...optimizing “cut-to-mutation distance”. Furthermore (3), we have developed a framework that, by combining...to mutation distance and editing efficiency.   3. Optimizing "cut-to-mutation distance" for homo or...or the other on each target site.   Figure 3: Optimal cut site to mutation distances for heterozygous...PMID: 23287722. PubMed Central PMCID: PMC3712628. 3. Paquet, Dominik, et al. "Efficient introduction of...mutations When thinking further about the distance relationship described above, we realized that, in addition...efficiency already drops by half and after only about 30 bp, it is no longer feasible to incorporate mutations...Additional Resources on the Addgene Blog Learn about High Fidelity Cas9 Variants How to Design Your gRNA...

...these programs typically consist of 1-2 mentors and 3-5 mentees who meet together for an 8 month formal ...cover during the year. Suggest that each person bring 3 topics and write them on a board to see which ones... a regular schedule might work. If not, perhaps a 3 hour breakfast once a month. All participants must...for Scientists Series. I have been thinking a lot about Mentoring for over 10 years. Many successful scientists...Medical School researcher Connie Cepko to learn about her mentoring style. While working on this project...postdocs I have started to talk to trainees more about peer mentoring. They want to find mentors to help... questions I get is, “What are we going to talk about?” There are an infinite number of ideas on the internet...

...the limit of what will be packaged. The 5’ LTR and 3’ LTR, respectively, act as RNA polymerase II promoter...are based on adenovirus 5 (Ad5).     Fig. 3 - Viral vector components of adenovirus Building...Proteins: Structure and Function. Viruses. 2021 13(3):388. DOI:10.3390/v13030388 Coffin J.M., Hughes S....aren’t reflected in the common components outlined above for gamma-retroviruses. While not required for lentiviral...and helper plasmids for all the viruses discussed above (gamma-retrovirus, lentivirus, adenovirus, and adeno-associated.... Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 1997. Principles of Retroviral Vector ...

...amounts to a small difference of two or three weeks. 3. How would you like to select for your flies? Sure...consumption. Drosophila are also used in cancer research (3). The sky is the limit. As mentioned earlier, even...PMID: 19563763. PubMed Central PMCID: PMC2753793. 3. V. A. Rudrapatna, et al. Dev Dyn. 241. 107–118 (2012...immunofluorescent images like in this paper (2). Metabolic assays such as Seahorse can be employed to look...Additional Resources on the Addgene Blog Learn about Using Cre in Drosophila Read Other Genome Engineering...

...measurements; (2) the cells you’re targeting; (3) how much expression is needed to read your experimental output...gene. Off-target expression can still occur (Fig. 3) - for example, some neurons can transiently express...; Taschenberger et al., 2017).     Figure 3: Comparison of two astrocyte selective promoters demonstrating...anterograde tracing system. Experimental Neurobiology, 29(3), 219–229. https://doi.org/10.5607/en20006 Rolland...Molecular Therapy - Methods & Clinical Development, 3, 16062. https://doi.org/10.1038/mtm.2016.62 Su, M....starting the selection process. Factors to think about are (1) your experimental conditions and measurements...

... transfer plasmid, (2) the envelope plasmid, and (3) the packaging plasmid. These are transfected into... virus in cell lines expressing galactosyl(alpha1-3)galactosyl (alphaGal) sugars were less stable than...with other viruses in a BSL-2 lab instead of a BSL-3 or BSL-4 lab. For more details, check out this review... spumaviruses to human serum by galactosyl(alpha1-3)galactosylation. Journal of virology 71:6174–6178 ... also toxic at high concentrations as described above and is inactivated by the complement system in serum...

...hydropersulfide-propagating enzymes 3-mercaptopyruvate sulfurtransferase (MPST) which catalyzes the desulfuration of 3-mercaptopyruvate...opioid use disorder,” says Hubbard. Figure 3: 20x confocal image of the periaqueductal gray (PAG...students as we have done in the past. Below, read about the five winners and their research.   Abhi Aggarwal...work has only been made possible due to the collaborative and highly interactive environment that Janelia...biosensors or biosensors that detect new redox metabolites are vital for researchers to better understand...

...-Integrin alpha V beta 3 [IPI-ITGAV/ITGB3.7] Integrin alpha-V,Integrin beta-3 Human, Mouse IgG1 Rabbit...-Integrin alpha V beta 3 [IPI-ITGAV/ITGB3.13] Integrin alpha-V,Integrin beta-3 Human, Mouse IgG1 Rabbit...roles of individual integrins. The Ha lab — in collaboration with the Springer lab, the Jaumouillé lab, the...recombinant antibodies and plasmids. To encourage collaboration and education in protein science, IPI hosts ...events and training courses on technologies and laboratory techniques related to antibody discovery. You...the Institute for Protein Innovation. She writes about all things IPI, from the scientists and scientific...

...CACCGNNNNNNNNNNNNNNNNNNNGTTTGGGTCTTCGAGAAGACCTATTCXXXXXXXXC -3’; Reverse oligo: 5’- AATTGXXXXXXXXGAATAGGTCTTCTCGAAGACCCAAACNNNNNNNNNNNNNNNNNNNC -3’, where ...PMID: 26780180. PubMed Central PMCID: PMC4744125. 3. Gilbert, Luke A., et al. "Genome-scale CRISPR-mediated...How Barcodes Can Be Used in Lineage Tracing Read about CRISPR/Optogentics Tools Resources on Addgene.org...

.... We have been using yeast models (3, 4) to answer questions about the evolution of the genes involved...complementation. In the procedure diagrammed in Figure 3, students first transform the S. cerevisiae met deletion...: US Government Office of Science and Technology. 3. Duina, A. A., Miller, M.E., & Keeney, J.B. Budding...Addgene Blog Learn about Synthetic Biology with BioBuilder Use Addgene Videos to Learn about Basic Lab Techniques...species, but these studies generally leave questions about gene function wide open. What if two seemingly similar...question, the plasmids are also used to teach students about restriction mapping and western blotting. Students...communicating science to the public. You can find out more about Clare at the Biology Dept. site.  References 1. ...

...acceptor fluorophore--think of it as a virtual photon 3. Relaxation of the acceptor fluorophore back to its...PMID: 27649177. PubMed Central PMCID: PMC5038762. 3. George Abraham, Bobin., et al. "Fluorescent protein...Nature 388.6645 (1997): 882. PubMed PMID: 9278050. 3. Meng, Fanjie, et al. "A fluorescence energy transfer‐based...can be done using FRET! Read on to find out more about this amazing imaging technique and find further ...our curated list of biosensors too, as another laboratory may have already created the sensor that you ...ability of labs to answer sophisticated questions about cellular processes. Currently, FRET experiments ... Tips for using FRET in Your Experiments Learn about Fluorophore Targeting for FRET Studies FRET Based...

...used outside of the laboratory. The detection threshold for these RNA sequences (3 nM), however, was quite... samples at clinically relevant concentrations (1-3 fM; 1x106-fold lower than before), and our combined...PMID: 25417166. PubMed Central PMCID: PMC4265554. 3. Pardee, Keith, et al. "Paper-based synthetic gene...allowed gene circuits to be deployed out of the laboratory in a cell-free and biosafe format that was stable... quite high and above what would be expected in patient samples (e.g.: blood, urine). Adapting toehold...

...    Step 3: Reviewing the plasmid summary on the Fragmid website.   Step 3 is simply a review...of emerging CRISPR technologies. Cell Genomics, 4(3), 100519. https://doi.org/10.1016/j.xgen.2024.100519...Destination vector (dropdown options depending on above selections) Module set (the set of components you...vectors) Target type (drop down options depending on above selections.) CRISPR enzyme (Cas9 [sp], Cas9 [other...

...Teacher, Vol. 79 No. 3, March 2017; (pp. 208-223). Link: http://abt.ucpress.edu/content/79/3/208 2. Fire, S....Nature, 391 (1998), pp. 806-811. PubMed PMID: 9486653. 3. Newmark, Phillip A., et al. "Ingestion of bacterially...experiments in the beginning. In most classrooms, the laboratory experience is focused on classical embryology... Biology with BioBuilder 3D Models for learning about CRISPR Resources at Addgene.org Find plasmids ...

...implement RSMs in E. coli with two plasmids (Figure 3 below): Input Plasmid: The input plasmid (high copy...and promoters with buffer sequences on their 5’ and 3’ ends (for insulation). A list of promoters and terminators...hammerhead ribozyme parts are described in Nielsen et al (3) and Lou et al (4). Despite choosing proper register...PMID: 25417166. PubMed Central PMCID: PMC4265554. 3. Nielsen, Alec AK, et al. "Genetic circuit design ...certain cellular behaviors, but little is known about the order or timing of these factors. Recombinase-based... three methods of state interrogation mentioned above (Sanger sequencing, qPCR, and fluorescent reporter-based...Bac Vectors Can Be Used for Genome Editing Learn about Michael Koeris' Journey from Grad Student to SynBio...

...covered (to avoid drying out) while 3 ml of melted agarose are mixed with 3 ml of 2X DMEM in a 50 ml sterile...easily achieved by splitting transfected cells into 3 plates at different densities for selection). A 2%...specific genes. Many years ago, people in the laboratory became frustrated with using glass cloning rings...the hood. A 48-well plate is also prepared with about 200 ul of culture medium containing serum in each...incubator, additional growth medium is added to about halfway up each well. Tips for using the agarose...with the firmer 2% agarose gel solution discussed above for picking. Many thanks to our guest blogger Iris...

...Examples of these approaches are given in Tables 3-7. 3. Making use of AAV’s selectivity for neurons: targeting...Learn more about retrograde AAVs Resources on Addgene.org Read our AAV guide Learn more about our viral...

...transgene contains a splice site acceptor followed by the 3’-end of the transgene. When a cell is transduced by...80% of muscle cells expressing mini-dystrophin.  3. Hybrid  Overview: The hybrid approach combines the...region of homology, a splice acceptor sequence, the 3’ half of the transgene. The full length gene is reconstituted...upstream of the 5’ splice donor and downstream of the 3’ splice acceptor of the transgene to help prevent ...first time AAV user Learn about how AAV is used in retinal gene therapy Learn about the parts of an AAV transfer...

...efficiency by approximately 15-fold (Chen et al., 2006). 3. Transduction efficiency depends on viral titer Both...approximately 20–50% of the packaging cells after 1–3 days of selection. A lower percentage of surviving...." Cell 113.2 (2003): 141. PubMed PMID: 12705861. 3. Chen, Sifeng, et al. "Freeze-thaw increases adeno-associated...grown at 30 °C instead of 37 °C. If you’re unsure about the integrity of your virus, perform a diagnostic...Additional Resources on the Addgene Blog Learn All about Adenovirus Associated Virus New to Virus? Check ...Check out Our Primer for Beginners! Learn All about Lentiviral Vectors Additional Resources on the Addgene.org...

...with us about adding new scientists to their labs. Read All the Posts in this Blog Series Our 3 PIs answer...'ve heard of labs where new hires spend the first 3 months reading and writing a proposal before even ...research question that people (especially you!) care about. Have you critically evaluated your plan? Have... to complete and has multiple benchmarks. Think about what your personal goals are and set milestones .... Take the time to generate new ideas and think about them critically before diving in. Research projects...