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  1. Viral Vectors 101: AAV Variables That Matter

    Type
    Blog Post
    ...primate. Likewise, in vitro efficacy doesn’t always translate to in vivo efficacy. You’ll therefore need to...Using AAV vectors in complex biological systems can be tricky at best, and downright infuriating at worst...site, or when using chemogenetic tools, where modification of cellular activity outside of your experiment... required is inversely related to transduction efficacy: the higher the efficiency, the lower the titer.... A. (2023). Various AAV Serotypes and Their Applications in Gene Therapy: An Overview. Cells, 12(5), ...Approaches and Further Perspectives of AAV Tropism Modification. Pharmaceutics, 13(5), 750. https://doi.org/...

  2. Plasmids 101: Dimers and Multimers

    Type
    Blog Post
    ...additional origins of replication compared to monomers, they are also able to replicate at a higher frequency...expected fragments for that plasmid. This phenomenon indicates the presence of a multimer.   Fig. 1:...the right, with the entire monomeric sequence duplicated in tandem, resulting in a plasmid that is twice...that affect the frequency of multimerization. Theoretically, as long as all the plasmid features remain ...other methods below should help in multimer identification.   Fig. 2: Gel image of Plasmid 61564...within a plasmid and, therefore, allow for the identification of plasmid multimers. Capillary Gel Electrophoresis...multimerization. One study by Bacolla et al. (2011) indicated that strain HB101 produced the highest yield of...

  3. Prime Editing: Adding Precision and Flexibility to CRISPR Editing

    Type
    Blog Post
    ...— prime editing typically needs to be optimized for each application, and the efficacy varies widely —... the indicated tool. ~ reflects an innovation that may optionally be combined with the indicated tool....also more precise. Base editors, for example, typically edit all the target bases within the base editing... target nucleotides are positioned within the canonical base editing window, base editing has higher efficiency...products to be indels while the efficiency of HDR is typically less than 10%. In contrast, the original paper...editing variants Four years after their initial publication, the Liu lab returned to the reverse transcriptase...C-terminal end of PEmax to produce PE7. PE7 significantly improves editing efficiencies compared to PEmax...

  4. Educational Planarian Tools for Teaching Developmental Biology Techniques

    Type
    Blog Post
    ...classrooms, the laboratory experience is focused on classical embryology techniques such as basic observation... in situ hybridization experiments. A recent publication [1] from the Sánchez lab describes the plasmids... manipulation. The technique involves using synthetically made RNA (riboprobes) with modified nucleotides... added to the tissue. The last step involves a chemical reaction to stain the cells that contain this ...easily generate off of a circular plasmid via PCR. Typically, to make these constructs, developmental biologists...the deposit) are described in the Sánchez lab publication [1] so that the students performing these experiments...target mRNA and silences its expression. More specifically, during the process of RNAi, an enzyme dubbed...

  5. Why Do I Need an MTA Anyway?

    Type
    Blog Post
    ...human subjects or clinical trials Acknowledgement in recipient’s future publications relating to the ...results, modifications, and inventions created by the Recipient Right to file patent applications on modifications... about intellectual property rights, pending publications, and commercial use often come up. An MTA can...pride in facilitating the exchange of not just the physical samples, but also the legal documentation that... Addgene has adopted the terms of the Uniform Biological Material Transfer Agreement (UBMTA) for its material...have opted into our eMTA system can even sign electronically or choose to auto-approve MTAs. Whatever your...

  6. Academic vs. Industry Postdocs

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    Blog Post
    ...setting—especially if job applications require teaching experience or high numbers of publications, which may take...their funding. Communication: In both industry and academia, you will have to communicate your science ...can be long—5 years or more, especially in the biological sciences—whereas industry postdocs have a set...course, you might have less time to explore scientifically since you can’t afford to spend too much time...Unless your academic lab is closely involved with clinical trials or has ties to industry, the drug discovery...people involved is staggering. The process is complicated. If you’re interested in the drug discovery process... proprietary compounds or information. Where publication is expected, the process is similar to academia...

  7. Multiplexed Capture of Promoter-enhancer 3D Chromatin Structures Using CRISPR

    Type
    Blog Post
    ...spatial and hierarchical organization of chromatin interaction networks  A second application for CAPTURE...created a dCas9-based CAPTURE (CRISPR Affinity Purification in situ of Regulatory Elements) method. The ...at once (Liu et al., 2020). CRISPR Affinity Purification in situ of Regulatory Elements: The original...but close together in terms of where they are physically located. DNA is then sheared into pieces small... original CAPTURE and CAPTURE 2.0 methods.   Applications of CAPTURE 2.0 CAPTURE 2.0’s increased rate ...identified with the multiplexed CAPTURE 2.0 largely replicated interactions previously identified with the original...CAPTURE 2.0 is the construction of spatial and hierarchical chromatin interaction networks. Think of these...

  8. Degrading DNA with Cascade-Cas3

    Type
    Blog Post
    ... as a helicase. Following recruitment by Cascade, Cas3 nicks the non-target strand. The helicase domain... Cas effectors to carry out genomic editing. Specifically, these types of CRISPR systems work using a ...recruit Cas3, which carries out the brunt of the mechanical work for editing. Class 1 CRISPR systems make...After these initial steps, Cas3 can perform its helicase and nuclease functions simultaneously, by continuously...debilitating disease! This is, of course, only hypothetical at this point. More time and energy must be ...Mashimo, T. (2022). Genome editing technology and applications with the type I CRISPR system. Gene and Genome...unidirectional genome editing in human cells. Nature Communications, 10(1). https://doi.org/10.1038/s41467-019-...

  9. Genome-wide Screening Using CRISPR

    Type
    Blog Post
    ...CRISPR screens check out Addgene’s 2016 publication “Practical Considerations for Using Pooled Lentiviral...experiments you use them for! A well-developed biological question and experimental system are absolutely...coli, and T. gondii genes.  2) What genetic modification are you trying to make? Addgene carries CRISPR...next-generation sequencing to check the quality of your amplification. If you've obtained a ready-to-use lentiviral... As such, sequencing CRISPR libraries after amplification and after a screen requires the use of next-... to skewed results. Selecting a cell type - Theoretically, any cell type can be used in a CRISPR screen...experiment, the use of appropriate controls, multiple replicates and several cell types can strengthen your results...

  10. CRISPR/Cas9 FAQs Answered!

    Type
    Blog Post
    ...with the newest CRISPR technologies and their applications can be exhausting. A quick search for "CRISPR...it's usually more efficient than plasmid donor. Typical design is to have 50–80 bp of homology arms on ...changes (>100 bp insertions or deletions), we typically use plasmid donor, with two homology arms on each...transfection in cells like HEK, we use around 400 ng of a typical size (~5 kb) donor plasmid with Cas9/sgRNAs vector...plasmids. For ssDNA oligo design, the Zhang lab typically uses around 100–150 bp total homology. The mutation... giving 50–75 bp homology arms. The Zhang lab typically use PAGE purified long oligos. For large changes...100 bp insertions or deletions), the Zhang lab typically uses a plasmid donor, with two homology arms on...

  11. Phage Directory: From Phage Therapy to a Repository of Phage Information

    Type
    Blog Post
    ...When Jessica Sacher, a microbiologist from the University of Alberta, saw that scientists were using ... request for phages to the directory does not typically come out until nearly the last minute when all...all other options have been exhausted. Theoretically, phage alerts can be sent out to the directory earlier...team must decide which labs should receive the clinical sample to test their phages. Because the sample...phages targeting organisms closely related to the clinical sample. For treatment, it is best to identify ...researcher may only know how to prepare phage for one application but not another or they may have only used phage...phage in basic research but not therapeutically: not all labs in Phage Directory have the resources or...

  12. CRISPR Protocol for Genomic Deletions in Mammalian Cell Lines [Video]

    Type
    Blog Post
    ...Regulatory DNA function is typically inferred based on correlative biochemical characteristics and ectopic...Canver of Boston Children’s Hospital and Harvard Medical School. Addgene is proud to present a video reprint... of Visualized Experiments (JOVE). The video publication by Stuart Orkin and Daniel Bauer's labs details... size and frequency [3]. There are several applications in which the production of deletions by paired...useful to study haploinsufficiency. Subsequent modifications at the remaining allele within the deletion ...for deletion screening by conventional PCR. Biallelically disrupted cells can be easily identified. Even...investigator at Boston Children’s Hospital and Harvard Medical School. His lab studies the molecular genetics ...

  13. SciComm: Taking Science to Elementary and Middle Schools

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    Blog Post
    ...my opinion, it was more important to communicate the applications of CRISPR over the details of how it...m a father of three, a scientist and science communicator, and I strive to be a volunteer and contribute...parents who can answer some of the surprisingly technical questions elementary students can ask. It doesn...unlike elementary schools, middle schools have dedicated science teachers. I tried contacting our school...simplistic and I would insult the students. Too complicated and I would quickly lose their interest. I won...great feedback from teachers and other science communicators.  What can CRISPR do? I spent more time on... dangers of people using the technology in an unethical manner, but I also tried not to be an alarmist...

  14. Deep Dive: Fixing and Permeabilizing for Immunofluorescence

    Type
    Blog Post
    ...fixatives - chemical cross-linkers and organic solvents.  Chemical Cross-Linkers Chemical cross-linkers...of Fixatives in Histology. Leica Biosystems. https://www.leicabiosystems.com/us/knowledge-pathway/fixation-and-fixatives...-linking cellular proteins. Cross-linking is a chemical reaction that covalently joins two molecules and...proteins can be lost during fixation with these chemicals, which impacts sample structure and could also... target to be washed away. Furthermore, these chemicals can also alter the structure of proteins, which...Tween-20, or saponin. Each detergent has different chemical properties that will impact the level of permeabilization... don’t even involve antibodies, they are still critical to the outcome of your IF experiment and deserve...

  15. Three Key Considerations for Precise Neuronal Targeting Using AAV Technologies

    Type
    Blog Post
    ...viral vectors (AAV), neuroscientists can combine classical genetic tools with the manipulation of specific...of these discussions as a publicly accessible practical guide which was recently published in Frontiers...of molecular tools and sensors Combinations of classical genetic tools, for example Cre/loxP or Flp/FRT...transgene expression by restricting expression to genetically defined cell types. Specific transgene expression...defined set of neurons can then be targeted with optical stimuli. The restricted light exposure to a specific...result in opsin expression in their cell bodies. Optical stimulation in a different brain area can then ... more versatile. To complement the wealth of genetically encoded tools available, researchers are also...

  16. Who Gives a Tweet? 9 Facts About Scientists on Twitter

    Type
    Blog Post
    ...scientists are overrepresented on Twitter Mathematical, life, and physical scientists are underrepresented. It...mostly because of this article: “A systematic identification and analysis of scientists on Twitter.” This...from the United States Standard Occupational Classification System  and Wikipedia. 2. Social and computer...Nature, Sciencemag, Wiley, Cell and Scientific American Figure 4: Top Scientific Domains Tweeted by Biologists...shown. Top: The follower network of life (green), physical (blue) and social (orange)  scientists largely...disciplines: social (left), life (center), and physical (right). How do you use Twitter as a scientist... Y., & Sugimoto, C. R. (2017). A systematic identification and analysis of scientists on Twitter. Plos...

  17. pSiM24: Simplifying Plant Genetic Engineering

    Type
    Blog Post
    ...for both transient and stable gene expression applications in plants. This smaller, easily customizable...Agrobacterium are termed binary vectors because they must replicate in two species: Agrobacterium and E. coli. Like.... coli. However, they also need to be able to replicate in Agrobacterium to ensure that they are maintained... Thus, binary vectors contain two origins of replication, one each for E. coli and Agrobacterium, along...bases each. By switching the E. coli origin of replication and codon-optimizing the Agrobacterium ORI, they...constitutively active M24 promoter has been modified with duplicated enhancer domains and characterized in studies...these properties of pSiM24 are suitable for many applications, the plasmid can also be customized due to its...

  18. Sonic Hedgehog? Sleeping Beauty? Learn About the Genes Behind Addgene’s Conference Room Names

    Type
    Blog Post
    ... well as neural development. Specifically, SHH is a morphogen, a chemical signal that diffuses down a ...exhibit abnormal segmentation during embryogenesis. Typically, regularly spaced denticles (bristles) outline...on the spindle of a spinning wheel but also a biological transposon system. Sleeping Beauty is a synthetic...transposon system including those from the original publication, Ivics et al 1997. Spaghetti Squash, a regulator...contract depending on the context. Contraction is critical for processes such as cell movement/motility, ...bunch of cookie monster eyes.”  Check out the publication to see for yourself. We have one plasmid in our...embryo (Azplazu and Frasch, 1993). Tinman is specifically involved in the formation of the heart amongst...

  19. Neuronal labeling with Spaghetti Monster

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    Blog Post
    ...controlled by collections of neurons that are intricately wired into circuits through synaptic connections...connections to that population. Limitations of common genetically encoded neuronal tracers Neuronal tracers such...neurons. As compared to epitopes tags, FPs are intrinsically fluorescent and can therefore be used in live.... Indeed, to create the smFPs Looger’s team strategically inserted 10 to 15 copies of single epitope tags...darkened chromophores. These probes are both intrinsically fluorescent and can be detected with highly ...photostable than GFP. In addition, antibody-based amplification of their signals is weak and increases background...postsynaptic cell adhesion protein that plays a critical role in neural development. It belongs to a huge...

  20. Twenty Years of Sharing: Expanding Accessibility

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    Blog Post
    ...range of logistical barriers. Addgene worked to identify, reduce, and remove these logistical barriers...’ve turned the available into the accessible. Physical access Addgene is located in the USA, but our ...Beijing Zhongyuan 2012 China Summit Pharmaceuticals International 2013 Japan Leehyo Bioscience... items to countries with a distributor. Arrows indicate the year we entered into an agreement with a distributor...Systems AAV Plasmids Blogs Origin of Replication What is a Plasmid? How to Design Your gRNA for...Digest Analysis We also introduced guides — practical primers to help users get started with tools like... country are represented in blue; darker blue indicates more views from that country.   While written ...
Showing: 481 - 500 of 1075 results