...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...

...[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...

...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...

... 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...not used for general plasmid selection. Instead, reporter genes are often employed to easily monitor transfection...expression levels in the cells. Ideally, the chosen reporter is unique to the cell, is expressed from the plasmid...assess transfection success. GFP is often used as a reporter and we will be covering this ever-versatile fluorophore...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...

...designed repeat protein combinatorial library by in vivo functional screen." Scientific reports 5 (2015...fusions reported in the literature include RNA detection, RNA cleavage, and translational repression or activation...of the many useful kits you can order from the repository: FX Cloning Kit BIOFAB Plasmid Set pCri System...including antibiotic efflux pumps and ankyrin seven-repeat proteins: Sjuts, Hanno, et al. "Molecular basis...strengths centered within GFP/RFP bicistronic reporters characterized in Cambray et al., 2013. These terminators... an Orthogonal Ribosome-Controlled Fluorescent Reporter."ACS chemical biology 11.1 (2015): 31-37. pCri...expression systems; the Cell-free expression test kit represents a set of 88 clones in a pCellFree vector that...

...Antibody Data Hub. Data reports  Each antibody submission is used to create a data report, which contains experimental...that information in the report as well.    Figure 3: An antibody data report, showing the results of ...methods section for the data report shown in Figure 3.   At the end of the report, you’ll find a results section... the data report shown in Figure 3. The antibody is rated "Pass". Curating the data reports Just like... page of the Addgene Data Hub.   Antibody data reports can be searched and sorted by gene/target, name...information is then formatted and published as a report with a DOI for citation. If you’re considering ...

...a subset of the new plasmids and viral preps in the repository through our hot plasmids articles. These...proliferate. Eastman et al. Cell Reports 2020. https://doi.org/10.1016/j.celrep.2020.107804 The CRISPR corner...to provide ready-to-use viral preps. Here are some of the new viral preps from recent months: The Dolcetto...Addgene Sc++ is a codon-optimized, engineered Streptococcus canis Cas9 nuclease that has a broad 5′-NNG-...inhibition pooled library is now available as lentiviral preps in addition to the pooled library format! The library...inhibits over 18,000 human genes. Find the lentiviral preps at Addgene hSyn1-SIO-stGtACR1-FusionRed is a Cre-dependent...channelrhodopsin. It’s now available as AAV5. Find the AAV prep here The red-shifted calcium sensor jRGECO1a is...

...invert the reporter and GeneR fragment. Then the second recombination event would excise the reporter from ...Spatio-temporal expression of optogenetic reporters: Since most optogenetic reporters are delivered using AAV injection...transduced reporter gene. Generating a FLEx switch to control expression of an optogenetic reporter would ...Cre-dependent reporter expression, the potential for background expression of the reporter will be greatly...shown in Figure 2, the cassette should contain a reporter DNA sequence (such as GFP) in the sense orientation...the two pairs of target sites. By flanking the reporter with one loxP site and one lox511 site in the ...would ensure that the reporter remains silent until a cell or tissue-specific Cre is provided. Since the FLEx...

... versions of this reporter to target the K9 (pcDNA3-K9 histone methylation reporter) and K27 (pcDNA3-K27...methylation reporter) positions on histone H3, which are known to be involved in repression and X-inactivation...The K9 reporter uses the HP1 chromodomain to recognise lysine 9 methylation, while the K27 reporter uses... promoter region of tumor suppressor genes can repress their transcription and inactivate them. Global... methylation in living cells. Essentially this reporter construct consists of one substrate (the histone-derived...methylation of these sites was controlled, the reporter consistently showed significantly different FRET...states. Thus, this system is a reliable tool in reporting methylation status of these sites. This new technology...

...epigenome may make certain processes, like cellular reprogramming of iPSCs, more efficient. In general, epigenetic...However, several proteins in the DNA oxidation and repair pathways work downstream of Tet1 to restore the...of Tet1 alone, or coupled with DNA oxidation and repair factors that allow for increased gene activation... other Tet1 delivery systems. Transcriptional repression DNA Methyltransferase 3 Alpha (DNMT3A) Vlatka...methylation is associated with transcriptional repression. Created with BioRender.com. DNA Methyltransferase...as a kit from Addgene.   Find Ready-to-Use Viral Preps for Epigenetic Modification Why use epigenetic modifiers...activates transcription, whereas dCas9-KRAB fusions repress transcription. Both of these methods also recruit...

...Traffic Light Reporter System: Can evaluate lentiviral component delivery as well as genome repair by non-homologous...in genome target sequence into EGFP reporter. Target DNA reporter system: Can evaluate component delivery...or knock-in a gene? Do you want to activate or repress gene expression? Are you trying to create a single...mutation? Do you want to create a fusion with a reporter protein such as GFP? All of these outcomes can... can be used in conjunction with activator or repressor domains to control gene expression. Base editors...detecting insertions/deletions using a mismatch repair assay? Or is PCR followed by gel electrophoresis...application. If you want to include a homology directed repair (HDR) template to create knock-ins, point mutations...

...primary antibody required. Reporters There are two main classes of reporters: chemiluminescence enzymes...are more consistent lot-to-lot. The highest reproducibility comes from recombinant antibodies (rAbs), which...that target the same protein — can be used to reproduce the benefits of polyclonal antibodies. Because...you need a specific conjugate, and changing your reporter molecule requires either re-conjugating or re-...-purchasing your primary antibody with the new reporter. Finally, each new conjugate will have to be tested...binding to your protein of interest. Protein preparation Before you can blot your proteins, you’ll need... is a sturdy membrane that can be stripped and reprobed multiple times. It doesn’t degrade during long-term...

...fundamentally the reporter construct monitors the expression of a nearby gene. The reporter gene serves as... screens to introduce reporters throughout an organism’s genome. These reporters have historically been...stimulate the expression of the reporter. The system is made up of a reporter gene flanked by a minimal promoter...promoter – all rely on the random integration of a reporter construct in close proximity to an endogenous ...example, if green fluorescent protein (GFP) was the reporter gene and green cells were observed in the kidneys... for with an uncharacterized gene. The actual reporter construct can be something phenotypic (such as...promoter. The system is composed of a promoter-less reporter which needs to integrate within an exon of a gene...

...RNA” base pairs to the repeat section of the crRNA, forming the repeat:antirepeat duplex. tracrRNAs also...Clustered Regularly Interspaced Short Palindromic Repeats. That contrast of simplicity and complexity is ...array. The CRISPR array is composed of identical repeat sequences and variable spacer sequences. The array...transcribed and processed into crRNAs, each including one repeat and one spacer. In bacteria, these crRNAs are bound... bound by Cas proteins (Cas9 shown here). The repeat sequence base pairs with the tracrRNA, and the spacer... of these arrays, with alternating spacers and repeats, was the original impetus for studying CRISPR. ...array, these sequences are called protospacers. Repeats: Bacterial DNA that appears between each spacer...

... materials in our repository. We started to increase accessibility to our repository by making it easier...Addgene opened in 2004, we focused on creating a repository that made it easy to share plasmids, both for...to — so far, we’ve shipped materials from our repository to an amazing 111 countries (Figure 1)! But while...that temperature-sensitive materials like viral preps and libraries were kept at the appropriate temperature...Systems AAV Plasmids Blogs Origin of Replication What is a Plasmid? How to Design Your gRNA for...Addgene blog in 2023. Views from a country are represented in blue; darker blue indicates more views from...YouTube channel, 2023. Views from a country are represented in blue; darker blue indicates more views from...

...the new plasmids, antibodies, and viral preps in the repository through our Hot Plasmids articles.  Here's...gene disruption in M. abscessus  Bioluminescent reporter of mechanical forces   Epitope tag antibody ...Western Blot for Anti-V5 [IPI-SV5-Pk1]. Addgene Report, https://doi.org/10.57733/addgene.l0ulu4. Hanke...through eight stages of embryonic development. Representative images of single focal planes through the middle...across species due to differences in natural DNA repair pathways and spontaneous mutations.  Here, the ...results in either bacterial death or indels after DNA repair. B) M. abscessus is first transformed with Cas9...an integrative plasmid containing tetracycline repressor TetR and resistance selection marker KanR. This...

...resistance gene and the origin of replication are necessary to replicate plasmids and make sure the cells... operon Inducible by arabinose and repressed catabolite repression in the presence of glucose or by competitive...transcribed promoters Inducible with galactose; repressible with glucose  Can be used independently or together...mRNA Yeast promoter for alcohol dehydrogenase I Repressed by ethanol Full length version is strong with ...expression Promoter from E. coli tryptophan operon Repressible Gets turned off with high levels of cellular ...lac operon Constitutive in the absense of lac repressor (lacI or lacIq). Can be induced by IPTG or lactose...expression. lacIq mutation increases expression of the repressor 10x, thus tightening regulation of lac promoter...