We narrowed to 972 results for: REP

...conjugated with a reporter or tag that can then be detected.  Typically, the reporter is an enzyme such...dish and incubate it with a reporter conjugated primary antibody. The reporter reacts with a provided substrate...simplicity we will discuss the more common enzyme-based reporter methods. For enzymatic signal detection, users... sandwich and competitive. In a direct ELISA a reporter enzyme conjugated antibody binds to antigen coated...antibody first binds to the antigen and then a reporter enzyme conjugated secondary antibody binds to ...detection antibody can be directly conjugated with a reporter enzyme or a conjugated secondary antibody can ...).  The sample (purple) is preincubated with a reporter enzyme labeled antibody and then added to the ...

...a subset of the new plasmids and viral preps in the repository through our hot plasmids articles. These...dCas-VPR repressor are introduced into the hPSC genome using AAV1 and contain an EGFP reporter. Expression...to provide ready-to-use viral preps. Here are some of the new viral preps from recent months: New GPCR...measurements of norepinephrine. These norepinephrine sensors include GRAB_NE1h (high affinity) and GRAB_NE1m...production in E.coli as simple as following a DNA miniprep protocol!  First, the authors created the pMAK...Expression of the dCas9-KRAB activator or dCas9-VPR repressor in hPSCs is also driven by a doxycycline inducible...promoter. The gRNA piggyBac plasmids include a mRFP reporter. With this system, they could quantify and track...

...validation carried out by the commercial repository. Repositories generally ensure cell line identity and...microbiology, and pharmacology (many of which are represented in Addgene's Special Collections). Arguably, ...invariably much older than those available from biorepositories leading to genotypic and phenotypic artefacts...interpretation in cell culture for this reason. Unreproducible results are rife. Misidentified or cross-contaminated...been known about since 1966 when Stanley Gartler reported that most of the 18 cell lines he had studied ...easier to conduct high quality cell culture. Biorepositories around the world ensure that the cell lines...The oldest, largest, and most diverse of these repositories is the American Type Culture Collection aka ...

...more efficient than homology-directed repair Homology-directed repair (HDR) stimulated by double strand breaks...during mismatch repair   ✓   ✓ ~ ~ ~ Transiently co-express mismatch repair inhibitor MLH1dn...originally published in Nature, is a “search-and-replace” genome editing technique that mediates targeted...heteroduplex is resolved by the cell’s mismatch repair system, and more recent prime editing innovations...innovations have improved the odds of mismatch repair favoring the edited strand. The first iteration of prime.... We’ll summarize the major ones in Addgene’s repository later in the post. Advantages of prime editing...relatively low, meaning that most Cas9-induced DSBs are repaired by non-homologous end joining. As a result, Cas9...

...biological replicates such as multiple mice in each group and C, technical replicates or repeated measurements...results.  Replicates Replicates are another critical control in biological experiments. Replicates can be...statistical power of an experiment. Technical replicates are repeated measurements of the same sample and are...experimental protocol. In the experiment above, you could repeat the tau western blot several times with the same...difficult to obtain meaningful results.  Biological replicates are distinct samples that are treated identically... on the results of a single mouse. Biological replicates help you to determine which responses arise from...will be used (Burry 2011). It does not need to be repeated for each experimental run as long as the protocol...

... Meghan Rego. Reproducible data are key to science, so scientists are used to repeating experiments to...the plasmid used to create the viral prep. In a clean viral prep, like AAV-44362 shown below, more than...confirm their findings. But no scientist wants to repeat an experiment because of poor reagent quality. ...the wrong plasmid? What if bacteria snuck into the prep during the production process? These questions are...contamination can be identified, and we will discard any AAV prep we suspect is contaminated. For AAV-68544, the best...method we wanted to see how low we could go. We prepared a range of spiked samples from 0.1-20% and discovered... detection software confirms the serotype of the prep by interrogating the VGS data for small predetermined...

...amplify and design primers that will bind to and replicate it. The following image shows the ends of the ...mutation than restriction enzyme based cloning. DNA replication by PCR has error rates that range from roughly...

...fluorescent reporter, directly couple silencing of your gene target to silencing of the reporter. This is...target sequence against the fluorescent reporter in an inverted repeat orientation. Therefore, a single hairpin...Figure 1). The plant kingdom has expanded its repertoire of Dicer enzymes and these different Dicer-like...desired target of RNAi is expressed as inverted repeats separated by a loop region that facilitates the...conclusion was based upon numerous phenotype discrepancies between gene silencing and knockout methods ... complementary to your gene target as inverted repeats (Figure 1). With this technique, multiple genes...fusing your gene target sequence to a fluorescent reporter sequence (discussed below). Examples of these ...

...suggested roles in transcriptional repression, activity dependent de-repression, chromatin remodeling, gene ...Programmable RNA Editing. Cell Reports 32:107878 . https://doi.org/10.1016/j.celrep.2020.107878 Sinnett SE, ...focused on therapeutic strategies that use gene replacement therapy, gene editing, MECP2 reactivation, RNA...RNA editing, RNA trans-splicing, and protein replacement to reverse the impacts of this disease.  As a...is a strong translational candidate for gene replacement therapy. In 2014, RSRT brought together a strong...have shown exciting and promising results for repairing mutations in models of neurological disease. Klaus... wild-type human iPSCs and then to successfully repair the mutation (Le et al., 2019). Beam Therapeutics...

...their relative representation in the edited pool. The black vertical dotted line represents the cut site...vertical black dotted line represents the cut site. Cutting and error-prone repair typically result in mixed...also provides a Knockout score (KO-score) which represents the proportion of cells that have either a frameshift...For a multiplex sample, ICE includes visual representations of all detected edit types in the sample. The...complete, a new screen appears with a graphical representation of the results and a list of the analyzed samples...used. Currently, ICE is configured for Cas9 fromStreptococcus pyogenes (SpCas9). ICE Score - The editing...correlation coefficient (r) is also computed and reported.  The higher the R2 value, the more confident ...

...must undergo homology-directed repair (HDR), a distinct cellular DNA repair pathway. Accomplishing this ...biallelic modifications. Most commonly, DSBs are repaired by the nonhomologous end-joining (NHEJ) pathway... convenient for generating gene knockouts, NHEJ repair does not allow introduction of specific sequence...involves simultaneously introducing a homologous DNA repair template, such as a sin­gle-stranded oligodeoxynucleotide... is relatively rare and DSBs are predominantly repaired by NHEJ. While other groups have focused on strategies...the same allele, probably due to concomitant NHEJ repair. At the PSEN1 locus, over 90% of the HDR edited...one Step 2 – Edit the cells a second time using a repair template that corrects the blocking mutation and...

...have newly-available AAV preparations added this year. We’ve added new viral preps of the dopamine sensor...2022.   Find iGluSnFR3 plasmids here! Find viral prep here: pAAV.hSyn.FLEX.iGluSnFR3.v857.PDGFR.codonopt... sustained deep-tissue imaging in vivo. Image reproduced from Liu et al. 2022, under CC-BY-NC license....license.   Find JEDI-2P plasmids here! Find viral preps here: pAAV-EF1a-DIO-JEDI-2P-WPRE and pAAV-EF1a-DIO-JEDI...activity of specific RTKs are limited. In a new preprint, Jared Toettcher and colleagues describe pYtags...of interest and co-expressing a cytosolic ZtSH2 reporter, they observe fluorescence localizes to the plasma...And orthogonal sensors with different ITAM/SH2 reporter pairs allow multiplexed measurement of different...

... and DNA repair. Black arrows indicate natural cellular processes. BER = base excision repair. Note: hypoxanthine...subsequently converted to thymine through DNA replication or repair. Fusing an inhibitor of uracil DNA glycosylase...the low editing efficiency of homology-directed repair (HDR), the pathway for resolving DNA double-strand...glycosylase (UGI) to dCas9 prevents base excision repair, which would change the U back to a C base. To increase...newly synthesized” to the cell. Thus, the cell repairs the DNA using the U-containing strand as a template...uracil N-glycosylase (UNG) during base excision repair. Adding a second copy of the UNG inhibitor, UGI... product purity, and these three improvements represent the fourth generation of base editors. Compared...

...[1]. In many tissues, stem cells serve to replenish/replace damaged cells that no longer function adequately...stem cells are: Their ability to self-renew or replicate while maintaining the undifferentiated “stem” ... location, mostly to maintain cell numbers or replenish dying cells. For example, hematopoietic stem cells...cells iPSCs are stem cells that are obtained by reprogramming differentiated adult cells by the ectopic expression...in 2006 in Shinya Yamanaka’s lab, where the reprogramming factors were delivered via retroviral infection...via iPSC formation, commonly referred to as reprogramming and, direct conversion from one somatic cell...: Implications for Regenerative Medicine and Reproductive Medicine. Geburtshilfe Frauenheilkd, 2015. 75...

...vectors are cloned and prepped in E. coli. However, they also need to be able to replicate in Agrobacterium ...plant infection, as well as plasmid yield during preparation in E. coli. Addgene depositor Indu Maiti has ...Agrobacterium are termed binary vectors because they must replicate in two species: Agrobacterium and E. coli. Like... Thus, binary vectors contain two origins of replication, one each for E. coli and Agrobacterium, along... the plant, similar to viral vectors’ terminal repeats. It’s a lot of information to fit into a single...bases each. By switching the E. coli origin of replication and codon-optimizing the Agrobacterium ORI, they...efficiency in both types of bacteria. They also replaced the promoter regulating the gene of interest with...

...unpublished materials. What about preprints? Addgene supports preprints and encourages authors to share ...alongside a journal article, a preprint, or without any paper at all. Posting preprints and openly sharing materials...transparency, support reproducibility, and accelerate science. We encourage preprint authors to deposit ...teams to keep searching, emailing, and waiting for replies. This updated policy will help our teams have clear...without a hold or to release the hold as soon as the preprint is posted in order to support these goals. And...article version any time later. We recognize that a preprint isn’t necessarily a clear sign that you are ready...your plasmids, so we won’t immediately release preprint-associated materials the way we will with journal...

...process by replacing the genes on the T-DNA region with our gene of interest or a reporter gene so that... transformation. Choose good reporter genes There are many great reporter genes available and which one... labor intensive, inefficient, and often not reproducible. Prior to creating transgenic cultures or plants...different treatments on transformation success. Some reporter gene examples are below. The GUS gene (β-glucuronidase...histochemical GUS-staining is a very useful visual reporter (Figure 2). The GUS enzyme cleaves a substrate...transformation success. Luciferases are great quantitative reporters and use a substrate to produce bioluminescence...half-lives then GUS and are therefore good temporal reporters to measure promoter activity, for example. Useful...

...targeting the pMB1 replicon on pTargetF (Jiang et al., 2015). In addition to the pMB1 replicon, pTargetF contains...In pCas9, we 1) replaced the promoter of gRNA-pMB1 with PrhaB, 2) changed the replicon of pCas into a ... the S. pyogenes Cas9, a temperature-sensitive replicon for self-curing, an arabinose inducible lambda-red... expression of the sgRNA that targets the pMB1 replicon, resulting in cutting of the plasmid. At this ... the gRNA on pCas that is specific to the pMB1 replicon of pTargetF may exhibit a more serious leaky expression...a non-temperature-sensitive replicon, and 3) added the sacB gene for counterselection to yield pEcCas....pEcCas. In pTargetF, we replaced the original 20 bp specific sequences on pTargetF with the ccdB gene containing...

... other before proceeding to library prep. Even with this “pre-prep” normalization, it is also often the...many labs. However, the high costs of sample preparation for high-throughput next generation (NGS) sequencing...enhancing the efficiency and simplicity of library prep. As part of our mission, we’ve been working with...Addgene to develop and apply our plexWell™ Library Preparation Technology for NGS-based sequencing and confirmation...Traditional NGS workflows are limited by costly library prep, as each sample requires an individual library. ...plexWell™ workflow is a sequential “pooled library prep”, in which a relatively large number of samples ...understated problem in multiplex NGS library preparation. The challenge here is that, as more samples ...

... (NM) 3' NNNNGATT  Streptococcus thermophilus (ST) 3' NNAGAAW  Treponema denticola (TD) 3' NAAAAC...their functions and simply called the repeated elements “direct repeats” and the unique stretches of DNA between... Species/Variant of Cas9 PAM Sequence Streptococcus pyogenes (SP); SpCas9 3' NGG SpCas9 D1135E... CRISPR arrays in bacterial genomes consist of repeated elements separated by unique sequences. When researchers...years of research, we now know that each direct repeat, combined with its adjacent spacer, ultimately ...ultimately encodes a single crRNA. The direct repeat regions contain sequences required for processing pre-crRNA...each individual crRNA. Bottom line, the “direct repeat region” combined with the tracrRNA forms the scaffold...