... mouse and rat lines. The Fenno Lab maintains a public folder (Link opens in a new window) of manuscripts...

...Depending on the research question and downstream application, one or several of those techniques can be used...your flow analysis. However, certain downstream applications, like an in vivo adoptive transfer of those ...available fix/perm kits allow for labeling of cytosolic proteins, as well as proteins of the secretory...intranuclear proteins of interest, you can label cytosolic proteins alongside the intranuclear ones. You ...You do not need to use a separate fix/perm cytosolic procedure in that case.     Figure 3: For intracellular...or free amines of proteins. Depending on the application, they can be membrane-permeant or membrane-impermeant...are positive or negative for the dye. The main applications of dye stains in flow cytometry are cell proliferation...

... ITRs serve as the origin of replication and are required for replication, packaging, and vector persistence...viral rep (replication) and cap (capsid) proteins, which bind to the ITRs and initiate replication. ITRs are...binding site for Rep78 and Rep68 to initiate replication and a terminal resolution site that serves as... Rep proteins are important for viral genome replication, these mutations can affect transduction efficiency... of research is important for downstream AAV applications.  Tips & FAQs Due to their high GC content and... and hairpin structures, ITRs are prone to replication errors and disruptive mutations and truncations...mutations?  During propagation, mutants rapidly replicate in a growth culture and result in a high percentage...

...what we do not understand, and the complicated and unique metabolic processes of plants are being investigated...involved in plant synthetic biology. Challenge #1: Public perception of synthetic biology One of the biggest...difficulties that plant biotechnologists face is public perception of genetic modification (GM) and synthetic...benefits and future potential of the technology to the public in an open, responsible manner. At Revolution Bio...obstacles to plant synthetic biology In addition to public resistance, an overall limited understanding of...knockouts to better understand developmental, metabolic, and photosynthetic processes. Often the tools... intellectual property concerns – patents and licensing agreements. Developing a product without the use...

...Require Sialic Acid Binding for Hemagglutination and Efficient Transduction but Differ in Sialic Acid Linkage...receptor Coreceptor AAV1 α2,3-/α2,6 N-linked sialic acids1 AAV receptor2   AAV2 Heparan sulfate...Laminin receptor (LR)3 CD93 AAV5 2,3-N-linked sialic acid4 AAV receptor2 Platelet-derived growth...vessel barriers. It is especially popular for applications targeting liver tissue. In addition, it has ...the blood-brain barrier, making it useful for applications targeting the CNS (Issa et al., 2023). Additionally...treatment, which can pose problems for gene therapy applications. Cross-reactivity of AAV serotypes varies by.... A. (2023). Various AAV Serotypes and Their Applications in Gene Therapy: An Overview. Cells, 12(5), ...

...constitutive splicing studies and has been optimized for high efficiency splicing. If intron splicing does not...potential applications of CRISPR/Cas9 in genome-engineering. One particularly attractive application involves...in order to avoid complicating effects from global toxicity due to loss of splicing. To this end, destabilizing... system widely accessible for many potential applications. Polstein LR & Gersbach CA, Nat Chem Biol ...amenable to both high and low throughput screening applications. The system can be readily modified to use different...cloning platform for imaging and flow cytometry applications Designing customized fluorescent reporters can...luciferase reporter system for detection of pre-mRNA splicing   The Gideon Dreyfuss lab has developed rapid-response...

...host genome, the virus is replication incompetent, meaning it can’t replicate and continue the infection... tools, thought was given on how to keep the replication of virus and its infectivity in check. Engineering...envelope plasmid), and the polymerase (Pol) for replication and Gag structural protein on yet a third plasmid... this safety feature prevents the virus from replicating and spreading in non-target cells, including ....    Viral cargo-specific safety   After replication competency, the second concern with virus safety... be disposed of following your lab’s biohazard policies. Administering Virus Many virus experiments involve... accident-prone researchers. Note that safety policies will vary slightly from organization to organization...

...between bacteria can also have serious implications for public health. Bacteriocin plasmids: microbial...us! A plasmid is a small piece of DNA that can replicate independently from its host’s chromosome. Although...Some bacteria possess plasmid-borne toxins like colicins and subtilisins to kill other bacteria in the ...plasmids carrying genes for bacterial toxins like colicin typically also carry the necessary resistance genes...survive their own secretions. Pro tip! Historically, colicin-producing plasmids have been referred to as “Col...inducible expression systems that allow tuning of metabolic pathways. Fertility (F) plasmids: microbial sharing... common plasmid features, such as origins of replication and promoters, check out our other Plasmids 101...

...most transposable elements (TEs) results in the duplication of this target site sequence at the site of insertion...This is similar to how retroviruses, like HIV, replicate. Class I TEs do not encode a transposase enzyme...enzyme.  Class I TEs are considered replicative since every time they jump they make a copy of themselves...retroviruses both in their structure and mechanism of replication. They contain two genes: gag and pol. The pol...they move. Most Class II transposons have a non-replicative “cut and paste” mechanism of transposition: they... it integrates, which produces a target site duplication (TSD). Created in BioRender.com.   Autonomous...transposition activity  Tol2 transposon plasmids    Applications of transposons Now that you’ve learned about...

...many CRISPR applications, and they’ve also had success in clinical gene therapy applications. Read on to...to learn more about the current (and future) applications of lentiviral vectors! Lentiviral vector history...original system, the possibility of creating a replication-competent virus via recombination between the...of recombination events necessary to create a replication-competent virus. Second, Tat is eliminated through...barcoding libraries have been used for complex applications like lineage tracing in heterogeneous tumors...developed integrase-deficient lentiviral vectors for applications that require the large genetic capacity of a...McGarrity GJ, Hoyah G, Winemiller A, Andre K, Stein D, Blick G, Greenberg RN, Kinder C, Zolopa A, Binder-Scholl...

...technique published every week! One of the newest applications is a colorful system that uses fluorescently...activity, catalytically dead Cas9 (dCas9) has many applications, the most well-known being transcriptional activation...characterizing the 4D nucleome. Future modifications and applications Ma et al. developed this technique using standard... et al. anticipate that the technique will be applicable to studies of cell cycle progression, epigenetics...to external stimuli. It could also have major applications to cancer, for example, the visualization of...Feb 23. Pubmed. Find the plasmids from this publication at Addgene. Dynamic imaging of genomic loci....2013.12.001. Pubmed. Find the plasmids from this publication at Addgene. A protein-tagging system for signal...

...Description Origin of Replication (ORI) DNA sequence which allows initiation of replication within a plasmid...plasmids are small circular pieces of DNA that replicate independently from the host's chromosomal DNA....virulence. All natural plasmids contain an origin of replication (which controls the host range and copy number...Minimally, lab-created plasmids have an origin of replication, selection marker, and cloning site. The ease... plasmids and the ability of plasmids to self-replicate within a cell make them attractive tools for the...plasmid by recruiting replication machinery proteins Antibiotic Resistance Gene Allows for selection of plasmid-containing...broadly utilize plasmids across a wide range of applications. Some common plasmid types include cloning plasmids...

...range of applications. Image reproduced from Torres Cabán et al. 2022, under CC-BY-NC-ND license.   Find...Image adapted from Ino et al. 2022, under CC-BY license.   Find MTRIA sensors here!   Ino D, et al. A fluorescent...They generated variants optimized for different applications (v82 has higher affinity for glutamate but slower...reproduced from Liu et al. 2022, under CC-BY-NC license.   Find JEDI-2P plasmids here! Find viral preps...ITAM to an RTK of interest and co-expressing a cytosolic ZtSH2 reporter, they observe fluorescence localizes...adapted from Farahani et al. 2022, under CC-BY license.   Find pYtags plasmids here!   Farahani PE, et...Image adapted from Koveal et al. 2022, under CC-BY license.   Find LiLac plasmids here!   Koveal D, et al....

...Ligation Independent Cloning (SLIC) in a previous Plasmids 101 post. Although SLIC may be more cost effective...assembly meets CRISPR Gibson can be adapted to more complicated cloning schemes, such as those where the vector...CRISPR technology and is described in a recent publication by the Lockey lab (Wang, et al. 2015). In this..., Gibson assembly improves on two aspects of the SLIC methods. First, it uses a dedicated 5’ exonuclease...is added to repair the nicks in vitro, whereas in SLIC these constructs are repaired in vivo, which ends...ends up being much less efficient. In addition to SLIC and Gibson, there are yet more homology based assembly...CPEC (circular polymerase extension cloning) and SLiCE (Seamless Ligation Cloning Extract) to name two ...

...to be doing in humans; We have not had a broad, public conversation or consensus around the ethics of ... us with the second critique. Absent of a broad public consensus on genome editing, there was a generally...revisited at meetings when sufficient time, additional public fora for data review, and more lab research could...could be used to bring scientists and the public together to build consensus guidelines, but there was ...t have to look far for proof that this model of public involvement, research, and consensus can happen... separate non-governmental groups with seeking “public views on emerging IVF techniques designed to prevent...recognize so-called bad actors among them and “self-police” or whether the media and academic conference response...

... a bacterial origin of replication (ORI) will not allow for plasmid replication in mammalian cells, for... their bacterial counterparts, including how replication occurs and whether selection is necessary for...the nuclear genome, in the absence of plasmid replication, the foreign DNA will be degraded or diluted ...always mission critical to make sure the plasmid replicates in the cell, as many of these experiments are...expressed in trans within the cell for effective replication. Cell lines expressing the Epstein–Barr virus...contain an ORI since the integrated DNA will be replicated as part of the genome. Because the foreign DNA...Antibiotic Resistance Genes Plasmids 101: Origin of Replication All Plasmids 101 Articles ...

...ITRs serve as a type of origin of replication to initiate replication of the dsDNA virus. Viral packaging...plasmid while the other plasmids with packaging or replication components are called helper plasmids. Below,... production, they can be helpful for certain applications and aid in ease of use. Our lentiviral guide...AAV are rep and cap. Rep is required for viral replication and also aids in infrequent genome integration...a helper plasmid and mediate efficient viral replication. More information on these viral genes is available...particles. E2A/E2B are responsible for viral replication, with E2B being the primary viral polymerase....is missing the endogenous E1 gene, making it replication deficient. This is typically supplied by the ...

...addition of a helper virus or plasmid allows it to replicate episomally, essentially remaining an extrachromosomal...extrachromosomal part of the host genome that can replicate and divide along with the endogenous DNA. AAV also...cell type doesn’t always lend itself to direct application of virus. However, the cargo capacity of AAV ...gamma-retro viruses. This is large enough for many applications, but in some cases multiple AAVs (co-infection...and AAV. If you’re wondering more about what applications these viruses are good for, we have plenty of...Adenovirus-Mediated Gene Delivery: Potential Applications for Gene and Cell-Based Therapies in the New...Gammaretroviral vectors: biology, technology and application. Viruses. 2011 Jun;3(6):677-713. DOI: 10.3390...

...enough to ensure the virus can replicate and spread. Instead, viruses replicate by hijacking the host’s system...low immunogenicity which makes them ideal for applications with whole organisms. Finally, viral delivery...delivery is typically straightforward across applications, while electroporation of a human, for example, isn...people in nature, extra safety measures, like replication incompetent viral particles, are required. We... (~4.5kb From basic science to clinical applications, viruses are true bench to bedside tools. Keep...our upcoming blogs on virus safety and viral applications to help you start (safely!) thinking about the... (or maybe first!) viral experiment?           Click Here to Find Viral Vector Resources at Addgene! ...

...thus allowing repair to take place before DNA replication. Indeed, they observed that 41/42 embryos were...attributable to CRISPR editing. Conclusions & implications One striking feature of this paper is the failure...consider that already-tested CRISPR alternatives are applicable in many situations. In the case of hypertrophic... need to be much, much lower before clinical applications could be considered. It is important to consider...consider the question of broader applicability of CRISPR germline editing. Nature News mentions that the...only way to answer questions of efficiency and applicability. Given moral and ethical concerns surrounding... biologists and non-biologists alike. Given the public significance of this research, we have the obligation...

...is useful for many metabolic engineering and synthetic biology goals. In metabolic engineering, the expression...problems. Rapidly dividing cells initiate genome replication more than once per cell division and thus, genes...genes closer to the origin of replication will be overrepresented compared to those further away. As a...or can interfere with the precision needed in metabolic engineering or other synthetic biology circuits...cells are dividing rapidly, multiple rounds of replication are initiated before the cell has divided. This...maximal gene expression near genomic origins of replication, the stabilized promoters created by Segall-Shapiro...the control of IPTG. Because RepA is a plasmid replication initiator protein, they could alter copy number...

...suitability of a systemic capsid for an individual application is dependent on a number of factors, including...test your chosen capsid before using it in an application.     Figure 1: AAV toolkit for targeted...doses are recommended for all systemic capsid applications. For example, AAV-PHP.eB is typically administered...experimentally determine the best titer for your application.   Pro tip!Want to know if a capsid is a good...always, validate the capsid for your specific application and animal model.  Other model organisms Tropism...capsids were deposited by the Gradinaru lab. For simplicity, in each section we’ll list the Gradinaru capsids...and AAV.MaCPNS2. Ex vivo macaque and human brain slices Several of the systemic capsids mentioned above...

...conditions.  Patents or Licenses If there are any patents or licenses that could prohibit Addgene...process. Figure 1: Begin the deposit process by clicking “Submit Online Deposit”or “Download Deposit Spreadsheet...name and match the name as it appears in its publication or as it is commonly known in your lab. Plasmid...descriptive purpose focuses on the practical application of the plasmid and will help a scientist outside...For plasmids that contain the R6Kγ origin of replication, choose the Pir1 strain. If your plasmid cannot... and add an explanation in the adjacent Patent/License Explanation field. Filling out and sending ...Distribution status for your plasmids: Hold for Publication or Distribute pending QC. The name of the Principal...

...able to replicate, and for a virus to cross a synapse from one cell to another, it must replicate. Modified...infects one cell (the “starter cell”). It then replicates within that cell, and nascent RABV cross synapses...infection occurs, subsequent cycles of viral replication and retrograde transmission occur, spreading ...limitations. First, these high levels of viral replication are toxic. Second, infecting subsequently higher...G-deleted RABV will infect the starter cell. RABV replication will then occur and the G protein (supplied in...order neurons, the G-deleted RABV can divide and replicate (since these processes are mediated by other viral...Wickersham et al., 2007).   Summary and RABV applications Use of G deleted RABV in conjunction with restricted...

...Ultramarine with hydrophilic amino acids to reduce non-specific interactions with cytosolic proteins. This...resources are limited or for bioart and teaching applications. Find a chromoprotein for your experiment! ...for specific purposes.     Chromoproteins for allelic exchange Figure 3: pTOX plasmids expressing...chromoproteins to create negative selection markers for allelic exchange. These vectors contain a toxin gene (regulated...-inducible promoter) and a chromoprotein gene. Allelic exchange occurs through two sequential homologous...and encode the tsPurple chromoprotein.  Find the allelic exchange vectors here! ShadowR, a novel FLIM-FRET... B. M., & Waldor, M. K. (2019). A New Suite of Allelic-Exchange Vectors for the Scarless Modification ...

...review how these indicators work, their general applications, and why viruses are essential to their use ...improvement. These sensors still have many useful applications, though, and if you’re in the market for a VSD...tissues depths (1 mm), important for certain applications such as in vivo tissue imaging.   Figure... detector in this system was dipicrylamine, a lipophilic compound that distributes itself within the plasma...sensing) hydrolyzes a precursor dye to make it more lipophilic, increasing its affinity for the membrane, where... fluorescent component (i.e., when the dye is lipophilic and integrates into the membrane), the voltage...your cell type of interest. For in vivo imaging applications, the ability to direct your voltage sensor selectively...

...lab while the publication page contains all the plasmids deposited from the same publication. Next up, we...through the depositing lab page, then onto the publication page, and finally on down to the individual plasmid...Great! Now, if I want to use this plasmid in a publication, how do I know who to acknowledge? Depositing...plasmids that are available through Addgene. Publication: If the plasmid has been published, the paper...the bottom of the page that help you cite this publication in your own work: How to cite this plasmid: The...depositing lab whenever their plasmids are used in a publication. Addgene makes this easy by providing a template...of our plasmid pages, and provides a list of publications which have used the plasmid (see our blog post...

...Fig. 2). Individual gRNAs are designed in silico using publicly available gRNA design software and synthesized...highlight some of the reagents that have been made publicly available through Addgene.  What makes CRISPR...over previous genome editing techniques is its simplicity and versatility. CRISPR/Cas9 consists of two ...essential for a given phenotype. Image Credit: www.flickr.com/photos/72005145@N00/5600978712/ What CRISPR-based...derivatives of Cas9. Several CRISPR libraries are publicly available through Addgene, with more being added...experiment, the use of appropriate controls, multiple replicates and several cell types can strengthen your results...information on CRISPR screens check out Addgene’s 2016 publication “Practical Considerations for Using Pooled Lentiviral...

...very little data on which might work in your application. It can be quite difficult to decide on which...be searched and sorted by gene/target, name, application, pass/fail rating, sample species, antibody species... how the antibody performed in the submitted application, along with a brief description of the experiment...experiment. If that data was included in a publication, you'll find that information in the report as well... section will vary slightly depending on the application — flow cytometry, western blot, immunohistochemistry...data showing how the antibody worked in your application. The submission process is easy, with simple,... through it at your own pace. To submit, simply click "Contribute Data."    Figure 6: Contributing data...

...in “crispr” and see what comes up. Before even clicking the ‘Search’ button a new content-aware suggestion...ll search for the calcium biosensor, GCaMP, and click through to the plasmid search results: More than... your lab. Instead of starting a new search or clicking through to a different part of the website, you...papers. Maybe they started their deposit prior to publication and were able to include specific plasmid ID ...terms we can use to find materials from this publication. Searching for the title of a paper works You...filters Not every plasmid search will start from a publication. You may have an idea for a plasmid that would...filters you do not need to start a new search. Simply click the ‘X’ next to any filter to clear it. Here’s ...