... event. What are exosome AAVs? In 2012, Casey Maguire and colleagues from Massachusetts General Hospital...that are released from cells during production (Maguire et al., 2012). They saw that these AAV particles...development of the exo-AAV isolation protocol by Maguire and colleagues, other researchers have tried to...delivery of therapeutic proteins to the brain which requires crossing the blood-brain-barrier. In summary, ...: 28082074. PubMed Central PMCID: PMC5368844.  Maguire, Casey A., et al. "Microvesicle-associated AAV ...

...that when you click on the link it will take you directly to the original article online. It is also designed...several journals or organizations now recommend or require authors to use specific PID formats. For example...Addgene registered prefix and the Addgene ID # are directly at the end of the n2t.net web address. For the...scicrunch.org/resolver/RRID:Addgene_87155) will take you directly to the lenti-Cas9-VQR plasmid page. Despite their...your choice. Or if in doubt, copy and paste the entire line. Cite plasmid using a resolvable compact identifier...

...thus inactivating genes), while breaks repaired by homology directed repair (HDR) pathways can modify the...causes two types of editing outcomes: Cas9 breaks repaired by the non-homologous end-joining (NHEJ) pathway...Richardson, Christopher D., et al. "Enhancing homology-directed genome editing by catalytically active and inactive...26789497.  2. Lin, Steven, et al. "Enhanced homology-directed human genome engineering by controlled timing ...

...scientists seek to directly probe and build new systems. Such goals require methods to directly activate and...TCS-induced responses are varied; one outcome is directed transcription from a given promoter. To harness...been greatly streamlined; only two plasmids are required per system. Applications of these systems The ...

... bacteria) or incorporate the genetic material directly into the genome to form a stable transfection....the plasmid, and can be assayed conveniently. A direct test for your gene of interest may be another method...viral-based ORIs to fill the void. These ORIs, however, require additional components expressed in trans within... Stably transfected cells may be considered an entirely new cell line from that of the original parental...important to determine the proper concentration required for efficient selection. This is usually achieved...

...use site-specific recombination, it also doesn’t require a ligation step. Based on the robust system of ...that position, preventing T4 from chewing back the entire 10–12 bases. As such, the use of LIC is often limited...the same tube. Alternatively, SLIC can also work directly on PCR products, with 5′ overhangs generated either...product with two 5′ overhangs, so these approaches require higher concentrations of insert to succeed. 5′ ...polymerase is much less expensive than the enzymes required for Gibson assembly. SLIC is a standardized method...

...though. Viral packaging and entry The genetic cargo requires viral packaging to infiltrate and integrate into...outlined above for gamma-retroviruses. While not required for lentiviral production, they can be helpful...packaging proteins for AAV are rep and cap. Rep is required for viral replication and also aids in infrequent...target different tissue or cell types. AAV also requires some back up from adenovirus genes – E4, E2a, ...LXs/Iva2 are primarily capsid assembly proteins required for packaging of the virus into particles. E2A...

...very similar for both, though tissue samples do require an extra homogenization step. For this blog, we're...throughout the sample processing and therefore will require crosslinking, which uses formaldehyde or UV light...provides a truly random digestion, but is harsher and requires more optimization. Enzymatic digestion, while ...both tissue or cell type, amount of protein, and desired fragment length for your output assay. A longer...https://www.wannapik.com/   Even though ChIP requires a larger upfront investment than many other antibody...

... is used by screen readers to provide visually impaired visitors with information about images that a ...primarily negative. Stupid, overwhelmed, discouraged, tired…words that indicated there were many barriers between...paper.  Of course, when you think about writing an entire scientific paper, you may feel it’s overwhelming...describe the image in a way that helps visually impaired readers receive the same information as sighted... writing. You can use them to help you write an entire manuscript, or you could use them to write alt ...

...for use in prokaryotes and eukaryotes, works via direct activation. In this system, the activator rtTA ...negative inducible pLac promoter. This promoter requires removal of the lac repressor (lacI protein) for...eukaryotic cell types (mammalian, yeast, plants, etc.) require unique promoters and there is very little crossover...induce a given promoter varies. Promoters that require only the addition of an external inducer, like ...very rapidly. In contrast, promoter systems that require transcription of a repressor/inducer will have ...

...often used for site directed mutagenesis. During this process, incorporation of a desired mutation into your... not the newly synthesized product from a site-directed mutagenesis experiment? The answer to both questions...not correct! Conversely, enzymes such as DpnI require methylation at their recognition sites in order...overlapping methylation Not affected Not affected DpnI Requires methylation for activity Not affected Not affected...

... and synthetic biology. The mistrust and anger directed at GM crops has burdened this emerging engineering...Revolution Bio, we are addressing this challenge directly by developing a plant biotechnology product specifically...through conventional breeding; synthetic biology is required to achieve this novel and unique phenotype. It...control over their interaction with it, we hope to inspire and open a new conversation about GMOs.  Challenge...technologies like those pioneered by Ewen Mullins of Ireland’s Teagasc.  Plant biotechnology is full of potential...

...plasmid(s). Fields marked with an asterisk are required. If submitting plasmid(s) using a spreadsheet ...Unpublished or pre-publication plasmids" and set a desired distribution status. You can submit "Published ...general description of the deposit. Select your desired distribution status: Hold for publication: we... need; you can copy and paste your information directly into the file. Email the completed spreadsheet... sequences cloned into it. gRNAs are used for directing CRISPR/Cas9 to a targeted genomic location, while...assembled or theoretical — it does not have to be entirely verified by sequencing. Upload any support files...

...’ve got you covered. Defining your control requirements To pick the best tool for the job, it’s important...important to know what the job requirements are. Here are the questions you should be asking. What kind ...recombinase. This is a time-tested system but does require knock-in of the LoxP sites, which is more difficult...control all the time If your experimental system requires tunable control - or maybe you’re just a control...time consuming. If your native protein doesn’t require this level of control, one of the previous options...

...that mismatch repair (MMR) interfered with the desired resolution of prime editing intermediates. Building... In PRIME-del, developed by Jay Shendure’s Lab, paired pegRNAs work with the PE2 enzyme from the Liu lab...that PRIME-del can be “multiplexed” using pooled paired pegRNAs.  Read the paper in Nature Biotechnology... strategy engineered by Wen Xue’s lab also uses paired pegRNAs targeting complementary DNA strands to ...sequence. As with other prime editing tools, the desired edits are templated by the 3′ extension of the ...

...retina, heart, and skeletal muscle. The dosing required to achieve these efficiencies was also between...mutation that causes Niemann-Pick disease type C, directly demonstrating the therapeutic potential for the...output the voltage changes in neural activity when paired with appropriate fluorescent optics. Existing red... sequences for genomic integration by homology directed repair. Using parts from this kit, the McCormick...The cofactor analog, phycocyanobilin (PCB), is required for PhyBΔ to be functional. Exposure to red light...

...cannot induce gene editing, but it retains its gRNA-directed targeting capacity. By using a gRNA to target ...of these orthologs is key: since each ortholog requires a different PAM sequence, a gRNA designed for ...different fluorescently labeled dCas9s are efficiently directed to the proper target sequence. They succeeded ...color is specified by PAM sequence, each color requires a different Cas9 ortholog, and the target sequence...in combination with gene editing. CRISPRainbow requires very short, 11-mer gRNAs, which do not induce ...

...scientists are acutely aware of these problems that directly affect their daily lives and future prospects....structure of the biomedical workforce. Quotes taken directly from participants (summarized in 2) touched upon...information on number of postdocs” “Postdocs are really hired to produce results, not scientists”  These comments.... Melanie Sinche is a STEM career counselor, Director of Education for the Jackson Laboratory for Genomic... at Scientific Meetings from Addgene Executive Director Joanne Kamens Learn how to be a successful manager...

...reviews our sequencing QC standards to ensure the entire team is using the same rubric and making the same...process also highlights other QC topics that may require additional attention during analysis. These include...and nucleotide populations. All of these topics require additional considerations to resolve. Sequencing...help QC scientists determine if enough reads were acquired from the MiSeq for assembly or with enough coverage...nanopore sequencing. This platform sequences the entire plasmid in one read and thus can resolve sequencing...

... allele decreases. Crossing mice to generate a desired phenotype Instead, genetic engineering should be...in the journey towards producing mice with your desired combination of mutations. Assuming your parental...generate mice that are homozygous for all of your desired genes. Remember Punnett squares from high school... are homozygous (or hemizygous) at all of your desired alleles to conduct your experiments. You should...target gene in your experimental mice. This will require several rounds of breeding in which you’ll want...