...University Cancer is one of the greatest examples of survival of the fittest. Cancer cells find a way to ... oxygen in the tumor, activates hypoxia inducing factor (HIF-1), which promotes the production of VEGF...effector cells of the immune system - cytotoxic T lymphocytes (CTLs), natural killer (NK) cells, and helper...increase angiogenesis around the tumor. This in turn regulates the expression of the tolerance-producing antigens...cells and T cells activated ex vivo are also contestants for cell-based immunotherapy. Using activated...Apoptotic and necroptotic tumor cells have also been tested as vaccines and therapeutics. Their uptake by DCs... patient deaths have occurred during CAR-T cell tests because treatment regimens were not chosen carefully...

...organelles with older (yellow to orange) protein. Image courtesy of Roberta Gottlieb. Laker et al., J Biol ...Your protein of interest does x, but it also participates in y. Depletion of your protein may upregulate...involving recombination elements such as FRT or Lox sites, and provides a modular system for easier exchange...known as SpyLigase and is a protein domain that promotes the formation of an isopeptide bond between 2 ... the event of a DNA break. Next, the gRNA being tested is expressed along with Cas9. If the gRNA effectively... 2014..) Our CRISPR-Cas collection of plasmids updates frequently, so visit our CRISPR pages often to ...Scientists can engineer unique enzyme recognition sites that flank a DNA module in an inverse orientation...

...Subscribe to CRISPR updates on our blog to keep current with the latest and greatest! Addgene resources...editing patterns with ssDNA repair templates. Their work demonstrates that different repair template strategies...out #KSgenome on Twitter if you missed our live updates! My most popular tweet from the conference is...for cellular HDR.   Having had time to digest my notes, since the conference, I think I may have finally...high on-target editing and low off-target editing rates across multiple systems. Read our introductory blog... been challenging, with protocols using dsDNA templates achieving ~1-10% efficiency. Easi-CRISPR uses ...

... plasmid, but are often seen in them.  Lox sites Lox sites, in combination with the Cre recombinase, are...two pairs of lox sites which are part of a dual lox system called FLEX or DIO. Lox sites are recognized ...using enzymes, like SmaI, which have recognition sites in the ITRs. Regular Sanger sequencing typically...and temporally control transgene expression. Lox sites are directional 34 bp sequences that can flank the...flips the sequence between the first pair of lox sites, and then excise the sequence between the second...transgene. By restricting the availability of Cre, lox sites lets researchers control the spatial and temporal...nuclear export of RNA and RNA translation, and promotes RNA transcript longevity. The polyA is located...

... is very suspicious! Refrigerate your plates: Placing plates at 4C for few hours after the initial overnight.... (To be completely accurate, IPTG binds and inactivates the lac operon repressor, thereby allowing lac...from the lacZ gene (a mutation called lacZΔM15) creates a non-functional β-galactosidase enzyme. Providing... rush the process: It is important to give your plates enough time for any intact β-galactosidase to be... and white colonies. Take care in making your plates: X-gal is light and temperature sensitive and needs...after autoclaving. If spread on top of pre-made plates, make sure it is evenly distributed and allow sufficient...of false positives: Blue-white screening only indicates the presence of AN insert, not necessarily YOUR...

...™ transposase facilitates the integration of the transposon specifically at ‘TTAA’ sites randomly dispersed...achieved using the piggyBac™ transposase, which facilitates direct integration of the donor-transposon (carrying...carrying your cargo of interest) into random ‘TTAA’ sites throughout the genome. When using piggyBac™, the...likely to be cell line-dependent, and should be tested in the cell line of interest. Importantly, by varying...significant rearrangement (such as when a stem cell initiates differentiation), transgene silencing may occur...the excision-only piggyBac™ transposase. Image courtesy of Transposagen.   How does piggyBac™ enhance...selection cassette by excision-only piggyBac™. Image courtesy of Transposagen.   How does the piggyBac™...

...that bind a specific ligand. Ligand binding then activates G-proteins to modulate downstream signalling. ...back-metabolized to clozapine and other clozapine metabolites and these can have off-target effects. More recent... allows for influx of Na+ and/or K+ ions, and activates neuronal activity. Figure 2. PSAM4-based LGICs... RHJ, DiBerto JF, et al (2015). A New DREADD Facilitates the Multiplexed Chemogenetic Interrogation of...

...lysine 27 methylation. When tested in living cells where methylation of these sites was controlled, the reporter...methylated states. Thus, this system is a reliable tool in reporting methylation status of these sites. This...a variety of diseases, ranging from cancer, to diabetes, to congenital disorders. A series of studies ...disease but increased their risk of death from diabetes. Conversely, paternal grandmothers who experienced...experienced in a period of life when the grandparents’ gametes were developing, an epigenetic change is thought... in the FRET signal. Subsequent demethylation separates the fluorescent units and so lowers the FRET signal...Bygren, and Soren Edvinsson. "Cardiovascular and diabetes mortality determined by nutrition during parents...

...400 vials in 35 minutes compared to a lab assisstant filling about 200 vials in 45 minutes by hand. How ...96 tips for easy sample transfer between 96 well plates. The second pod can pick up 8 tips and position...useful if you’re transferring samples from 96 wells plates onto agar q-trays where the samples must be spread...BIOROBOT Universal Platform is ideal because it eliminates the potential of human error or cross contamination...this automated protocol, we can prep two 96 well plates in just under 3 hours or one 96 well plate in about...uses it to fill as many vials as it can in the shortest amount of time. Racks of 48 vials are set up on...using electronic multichannel and single channel pipettes. These can speed up any project in which you need...

...Thursday, or, at the latest on Friday. It’s best to split cells before the plates are more than 80-85%...air and beyond Turn on the hood for at least 15 minutes before you start any tissue culture work and make...Also, avoid unnecessary talking because talking generates microbe-laden aerosols that may enter the hood...70% ethanol immediately. Finally, avoid sticking pipettes into solution bottles directly, rather decant ...they are passaged. This could change their growth rates and morphology, rendering their continued use inadvisable...Anything between orange and light orange usually indicates that the cells have grown enough, are making their...Benjamin Franklin)! It’s good practice to label your plates and/or flasks with your name, date, the cell line...

...so far! Why do we need PAM flexibility? IDT estimates that there is an NGG PAM every 42 bases in the...might be undesirable due to potential off-target sites or low predicted on-target efficiency. In cases ...aureus and N. meningitidis require different PAM sites than SpCas9, in this case, NNGRRT and NNNNGATT, ...activity on non-NGG PAMs, generating xCas9 3.0-3.13. To test xCas9 3.0-3.13, Hu et al. first restored catalytic... that the clones were capable of cleavage, they tested the clones against an NNN PAM library in bacteria...editing rate as SpCas9 at an NGG PAM. When they tested a variety of NG, GAT, GAA, and CAA PAMs, they saw...outperformed SpCas9-VPR in activating transcription. Sites containing NG, GAA, or GAT PAMs averaged 56-91% ...

...and genetics. She loves to communicate the latest and greatest in scientific content.      References 1....ready for purchase. But what is the easiest and fastest way to use the system in mice? We'll have a post...either scan the genome yourself for potential gRNA sites, or you can utilise several online tools that do...supplied by the Zhang lab. This simple website invites you to input the target region (approximately 500bp...plasmid using standard cloning techniques (The XX indicates where your target is inserted, disrupting the ...details on this plasmid, see Mashiko et al., 2013). Testing the efficacy of your gRNAs is then a simple matter...of gene modification in vivo. Find Plasmids for Testing Your gRNAs in vitro at Addgene CRISPR/Cas9 gene...

...be activated from low fluorescent states to high fluorescence states, they can change from one fluorescent... light. The fact that blue light both activates and inactivates Dronpa is not ideal, since this means ...488nm blue light. The T203H mutation in PA-GFP generates a chromophore population that is more neutral,...but then further exposure to blue light also inactivates the chromophore, leading to a non-fluorescent...photoactivation. A high contrast ratio usually indicates that a PA-FP has a low level of spontaneous fluorescence...and display bright and photostable green and red states. Beyond these imaging applications PA-FPs have ... the National Academy of Sciences of the United States of America 101.45 (2004): 15905-15910. PubMed PMID...

...create notes directly from webpage clippings, photographs, and file attachments. Organize your notes into...was in one of those four white binders. Thirty minutes later you’re frustrated and no closer to starting...pages in your notebooks and go wild with sticky notes and labels. Color code your Post-its and write brief... brief descriptions on each. Place the notes sticking up out of the top of the notebooks, so that you ...notebooks, create checklists that link to other notes, and use the search bar to quickly find what you...the protocols, guides, or references that your labmates ask to borrow most frequently. Copy these into...the revision history. Now you’ll be able to make updates to protocols in real time. You may also want to...

...low-affinity sites, the proteins work cooperatively to separate nearby AT-rich regions of DNA. In a test tube,...Then, the ATP-bound DnaA binds multiple specific sites within the ori called DnaA boxes. These boxes come...controlled plasmids. Binding of IHF (green) redistributes DnaA (red) from binding primarily high-affinity...helicases to continue unwinding the strands. This creates two replication forks that move in opposite directions... to regulate DNA replication. DnaA negatively regulates its own expression, so high levels of DnaA act...methylating newly synthesized DNA, blocking binding sites, or trading out ADP for ATP to re-activate DnaA....binding and contributing to DNA unwinding. This promotes initiation at the plasmid ori, after which replication...

...recombination sites. These sites are target sequences of PhiC31. In the SPARC system, attP sites are located...-UAS system along with precisely truncated attP sites to control recombination of the gene construct. ...was developed by Thomas Clandinin’s lab, and incorporates several common genetic tools in Drosophila (...labeled. PhiC31, a recombinase. It specifically integrates an attP site into an attB site. In the SPARC ...irreversibly recombines one of the two competing attP sites to drive expression of either a stop cassette or...used SPARC to image the calcium response in the dendrites of T5 neurons by placing the calcium indicator...

.... Polyclonal antibodies, which bind to multiple sites on an antigen, are cheap and can help amplify a ...reporter. Finally, each new conjugate will have to be tested to ensure it doesn’t impact the antibody’s binding...400 kDa MES for proteins <40 kDa 180 V, 30 minutes Good shelf life; runs at neutral pH (reduced ...400 kDa MOPS for proteins > 30 kDA 200 V, 30 minutes Good shelf life; runs at neutral pH (reduced ...your membrane. It requires anywhere from sixty minutes to overnight to run, and is recommended for very...proteins. A semi-dry transfer is faster — 3–30 minutes — and uses less buffer, but it is not as efficient... These proprietary systems are fast (often <10 minutes) and simple, utilizing individually packaged membrane...

... AAV variant, rAAV2-retro, that migrates retrogradely at higher rates than any other AAV serotype (Tervo...neurons. Neurons are composed of a cell body, with dendrites receiving incoming information, and a projecting...at the terminals of axons form connections to dendrites of proximal neuronal cells. In the specific example...anterograde transport relies on kinesin. Different rates of speed for various forms of retrograde and anterograde...infection to the central nervous system where it replicates, spreads, causes neurotoxicity, and is lethal...et al., 2008). Herpes Simplex Virus (HSV) also migrates through neurons where it can replicate and by ...anterograde tracer and injected at several hundred sites across the entire mouse brain. Traced neurons expressed...

...landing sites,” then the recombinase handles the installation of the donor sequence. The latest work evolves...epigenetic silencer Recombinant antibodies for marking astrocytes MagIC-Cryo-EM for sample enrichment and structure...PRC2 with RvLEAMshort (1:6 molar ratio) with 10 minutes of glutaraldehyde crosslinking: representative ...sequences containing attB/P recombinase landing sites on opposite DNA strands. When the prime editor releases...sequence with the attB/P landing site. A recombinase integrates a sequence from a donor plasmid containing a ...resulting in the integrated sequence between two attR/L sites. Image reused from Pandey et al. 2024 under CC-BY...autoinhibition and stimulating the methylation of nearby CpG sites on the target DNA, and thus inactivating the promoter...

...Addgene. What you might not know is that Addgene distributes curated collections of plasmids as kits with ...pExpression vectors) plasmids. The type IIS restriction sites used in these vectors are of low abundance (meaning...yeast, mammalian cells, insects, and even Xenopus oocytes. The expression vectors additionally contain various...protein tags (His, Avi, and GFP) as well as protease sites to facilitate protein purification. We hope this... Using a variety of GFP and RFP fusions as their test genes, the researchers at the BIOFAB showed that...peptides to facilitate protein folding and/or protease sites to remove the epitope tags. Different plasmids in...library of eGFP-tagged human ORF clones to allow testing and comparison of different cell-free expression...