...that stably express a gene of interest. While the general approach for using lentivirus, infect and select...consider is the age of your culture post thaw. In general, the higher the passage number of a culture the...routinely test for mycoplasma contamination. There are several commercially available mycoplasma detection kits...parameters. For example, while freeze thaw is generally not recommended, Holic et al. found that lentivirus...your target cells Labs tend to follow the same general procedure for lentiviral infections, produce the...earliest steps in lentiviral transduction is the interaction between the viral envelop and the receptors on... on the target cell surface. The possible interactions and hence possible targets for a lentivirus are...

...enhancers, and cross-cassette interactions in gene expression, and to generate multi-gene knockouts. Mammalian...MoClo background MoClo is a clever way to assemble several DNA parts in a specific orientation using Type ...2011). It can be performed at multiple levels to generate anything from individual transcription units (...products without the undesirable side products generated during chemical synthesis of these molecules. ...bacteria. If your yeast experimental goals are more general in nature, or you aren’t planning on using K. phaffii...Multiplex Yeast Toolkit builds on the YTK and adds several additional capabilities, including conditional ...., 2014). This kit is a great starter for your general plant cloning experiments. Addgene also has two...

...photobleaching. Interaction with receptors: Understanding how fluorescent dyes interact with specific receptors...intermolecular FRET for ligand-receptor interactions, require specific considerations regarding the choice of fluorescent...crucial in the world of biological research, medical therapy, and even drug development (Grimm & Lavis, 2022...targets through fluorescent labeling. There are several advantages to using these self-labeling proteins...is bound through green light emission. Key considerations in selecting fluorescent dyes/ligands Effects...best practice to determine optimal DOL is through several small-batch experimental labeling. Size is also...under certain conditions, which is an important consideration for assay design (Breen et al., 2016). When ...

...in Steve Pollard’s lab at the MRC Centre for Regenerative Medicine (CRM), Edinburgh. Most commercial antibodies...therefore, critical to understand how proteins interact with one another (or with DNA) to perform diverse...the most popular approaches to study protein interactions is the pull-down assay, wherein a protein of...analysed by mass spectrometry to identify the interacting proteins. Similarly, in ChIP-seq assays, chromatin...however, to these IP-based assays is to find (or generate in-house) a pre-validated antibody for each target...throughput because numerous cell lines can be generated, each with a different tagged protein of interest...these factors prevent stem cell researchers from generating clonal knock-in lines, especially for ambitious...

...Chamberlain JS (2011) Gene Therapy of mdx Mice With Large Truncated Dystrophins Generated by Recombination Using... reassemble fragmented genomes is the basis of several split vector approaches developed in the early ...full-length transgene (Chamberlain et al., 2016). It’s generally accepted that some form of homologous recombination...exact mechanism is not yet known. Drawbacks:  Generally an overlapping split vector has equal or higher...is currently no one ideal split vector system. Overall the efficiency of transgene expression from split... of Adeno-Associated Virus Capsids. Human Gene Therapy Methods 27:1–12 . https://doi.org/10.1089/hgtb.2015.140...Capacity in a Transgene-independent Manner. Molecular Therapy 16:124–130 . https://doi.org/10.1038/sj.mt.6300322...

... for Autoinhibition Release of Methyltransferase (CHARM), with several advantages over the earlier system...and stimulate the endogenous methyltransferase. The strategy is generalizable to a variety of DNA-targeting...Arimura et al. used MagIC-cryo-EM to determine several structures (at < 5 Å resolution) of the linker ....    Figure 2: A) When proteins (blue) interact with the air-water interface (AWI) before vitrification... 2024). The best-performing variant yielded an average integration efficiency of 23%, a more than four-fold...editor consisting of dCas9 fused to a DNA methyltransferase — that can drive lasting transcriptional silencing...  Rather than overexpressing an exogenous methyltransferase for epigenetic silencing, which can be toxic...

...mouse inner ear. Fourth-generation cytosine base editors The fourth-generation base editors, BE4, reduce...variety of targets in human cell lines, with an average indel frequency of only 1.1%. These numbers are...over Cas9-mediated HDR for the loci tested, where average HDR-mediated editing frequency was only 0.5%, and...18 bases upstream of the PAM.  David Liu’s lab generated BE3 variants with other deaminases: AID, CDA1,...these three improvements represent the fourth generation of base editors. Compared to BE3, BE4 offers ...ABE7.10 is the most active editor, displaying an average editing efficiency of 53% with an editing window... phage-assisted evolution selection systems to generate ABE8e(TadA-8e V106W), which edits ~590-fold faster...

...organism. These features make AAV ideal for human therapies and in vivo laboratory experiments ranging from...terminal repeats – the sequences that flank the deliverable DNA) are removed, resulting in recombinant AAVs... over 4.7 kb. Co-infection rates for AAVs are generally high enough for this approach to work, although...some mammalian tissues has shown expression for several years (Wonjo et al., 2013). Remember that AAVs ...As a starting point, we recommend checking the literature and our AAV data hub to see if others have used...transduction independently of DNA synthesis." Gene therapy 8.16 (2001). PubMed PMID: 11509958. Vandenberghe...adeno-associated virus manufacturing." Human gene therapy 21.10 (2010): 1251-1257. PubMed PMID: 20649475....

.... To overcome this limitation, we created a generalizable tag-based strategy, which we refer to as the... target degradation (Figure 1). Plasmids: We generated a series of plasmids that are available from Addgene...of N- or C-terminally FKBP12F36V-tagged fusion chimeras or locus-specific knock-in of FKBP12F36V. Detailed...or Jay Bradner ([email protected]). Considerations when getting the dTAG system up and running...your lab Confirm the functionality of your fusion chimeras For each protein-of-interest, it is important ...FKBP12F36V tag without disrupting protein function. A literature search can help determine which terminus is favorable... evaluate degradation of the FKBP12F36V-fusion chimera of interest. In the cell lines we have evaluated...

...Fragments generation After you’ve designed your cloning scheme, you’re now ready to generate fragments... specific, there are few general parameters that you can adjust: temperature and time of incubation, and...fragments. You can generate the parts of DNA that you want to assemble together in a combination of different...multiple digestion be sure that the buffers and temperature are compatible between the different enzymes....genome organisms like mouse or human. There are several online tools that can help you with that, they ...companies selling PCR reagents. Nowadays commercial polymerases are very efficient, but if you are having difficulties...conditions. Purifying DNA fragments Once you’ve generated your DNA fragments, it is always a good habit ...

...separation is maintained by 2 sets of interactions : 1. Interactions between the non-target strand and a... through interactions between the non-target strand and the HNH/RuvC groove, and interactions between ...weakening interaction sbetween the non-target strand and Cas9, Kleinstiver et al disrupted interactions between...by the Cas9 HNH and RuvC nuclease domains 2. Interactions between the target strand and the gRNA (see ... the HNH/RuvC groove, then they could weaken interactions between the groove and the negatively charged...that if they could weaken sequence independent interactions between Cas9 and DNA, then they could diminish...effect on specificity was similar; SpCas9-HF1 generated fewer off-target cuts when compared to WT SpCas9...

...to base transition editing, we can expect several generations of optimization before the field begins to...review how CRISPR-derived base editing works in general. Base editors use either catalytically dead Cas9... Beyond UNG: Optimizing C → G editors Now that several labs had shown that preferential C → G editing ...rXRCC1, or the DNA binding and lyase domain of DNA polymerase β (PB) (Chen et al., 2021). Both of these fusion... Sun and Erwei Zuo labs tested and implemented several of these advances for CGBEs (Yuan et al., 2021)...efficiency and produced fewer indels than first-generation editors. This team also demonstrated that different... proteins whose fusion improved the CGBE: DNA polymerase D2 (POLD2), exonuclease 1 (EXO1), and RNA binding...

...Tethering dCas9 to the histone acetyltransferase p300 or the DNA methyltransferase DNMT3A allows researchers...2013). Enzymatic domains tethered to dCas9 often interact with DNA independently of dCas9 targeting (Galonska... independent of their enzymatic activity. They interact with transcriptional machinery to modify local...modifying other sites in the genome and directly interacting with transcription machinery.   Well, I’...demethylation?  Dividing cells depend on DNA methyltransferase 1 (DNMT1) to copy methylation marks from parental...   Let me leave you with a few practical considerations: Design your gRNA to overlap the CpG of interest...that unmethylated CpG so that nothing else can interact with it. Remember, the goal of this experiment...

...demonstrated that the caffeine concentrations in several beverages could be estimated by measuring growth of ...permits it to bind to the CIB1 (cryptochrome-interacting basic-helix-loop-helix) protein. After fusing...proteins of interest, a researcher can cause them to interact by stimulation with blue light.  Similarly, light...work including the soCoChR-GFP plasmid as well as several derivatives encoding alternative promoters and/...as starting material for the bioproduction of therapeutic methylxanthine drugs like theophylline. To facilitate... required enzymes, including a glutathione S-transferase homolog from a Janthinobacterium sp. Moreover...minimal media supplemented with various caffeinated beverages, but not with caffeine-free drinks. Finally, they...

...things) gene delivery, generating pluripotent stem cells, and developing gene therapies for monogenic diseases...excellent tools for genetic engineering. There are several different viral vectors to choose from, for example...which includes the gamma-retrovirus and lentivirus genera, have the unique ability to integrate permanently...separating viral genes and transgene expression across several plasmids. The most commonly used viral vector systems...of the lentiviral elements and the 2nd and 3rd Generation Lentiviral Systems can be found in our Lentiviral...gamma-retroviral packaging plasmids are not interchangeable. General envelope plasmids, such as VSV-G, may be used across...interest into this tetracycline inducible, 3rd generation lentiviral vector backbone from the Shih lab....

...structural determinants of protein-DNA interactions and how these interactions regulation gene expression. Recent...structural determinants of protein-DNA interactions and how these interactions regulation gene expression.     ...are being awarded free plasmids form Addgene to accelerate their research. Read on to learn how these researchers.... These studies are expected to identify new therapeutic targets/solutions and enable scientists to discover...mutations. References: Baldwin, Enoch, et al. "Generation of ligand binding sites in T4 lysozyme by deficiency-creating...in brain development since 2000 and have shown several mechanisms by which they affect brain and muscle...muscle development. The Gronostajski lab has generated mice wherein it's possible to delete the functional...

...needed for optogenetics. There are several important considerations with fabrication of the fiber optics...pulse generator such as the A.M.P.I Master-9 Pulse Stimulator, which can control output of several lasers...equipment, most of which is relatively cheap). Several protocols have been written outlining the different...stimulation. The scatter effect is also an important consideration when preparing the implant itself, which brings...least 85% of the initial laser output. The same considerations apply to the polishing and preparation of the... channel, Channelrhodopsin 2 (ChR2), exists in several states: closed/light receptive, open, closed/light...manipulation of the laser power source itself. Pulse generation (TTL pulse) is a standard electronics protocol...

...your serotypes, titer, and promoter. Note that several steps in testing are effected by multiple AAV variables... as proxies.  Finally, the size and receptor interactions of a serotype can affect spread from the injection...and are useful if you're going to be working in several different systems or want to transduce a large ...Various AAV Serotypes and Their Applications in Gene Therapy: An Overview. Cells, 12(5), 1–41. https://doi.org... Gene Delivery to the Hypothalamus. Human Gene Therapy, 21(6), 673–682. https://doi.org/10.1089/hum.2009.169...Carroll, S. J. (2019). Astrocyte-selective AAV gene therapy through the endogenous GFAP promoter results in... in the rat spinal cord following injury. Gene Therapy, 26(5), Article 5. https://doi.org/10.1038/s41434...

...cloning. The general cloning strategy described in the associated publication can be used to generate any combination... a modular mini-Tn5 vector that can be used to generate random mutagenesis libraries or to insert heterologous...oxidized. The Timer tool has already been used by several groups to monitor protein and cell ageing, as the...action via sequestration. The strategy relies on several observations: Rapamycin is a membrane-permeable...vectors are used with Multisite Gateway cloning to generate the final construct. Up to eight entry vectors...demonstrated the use of the SpyLigase peptide-peptide interaction to link affibodies or antibodies against common... Despite these advances, the Ting lab’s first-generation APEX suffered from inconsistent activity levels...

... plasmids for generating your own libraries are also available in the Sybody Generation Toolbox plasmid... two different coronaviruses into a llama over several days and isolated special camelid antibodies that...proteins that may unfold due to camelid high body temperature (37.2 - 39.2°C). The Seeger lab found a way around... around this by using plasmids to generate sybodies, synthetic nanobodies against the spike protein (Walter...blog post.  A rapid selection platform for generating sybodies against membrane proteins Developing...membrane protein of interest using the Sybody Generation Toolbox and commercially available reagents in...libraries (Zimmermann et al., 2018). Find the Sybody Generation Toolbox! Starting with libraries and a biotinylated...