... integration of a novel gene randomly into their genome, some transgenic strains are difficult to breed...expression level of the transgene. In existing strains of mice, the transgene can be maintained on a hemizygous...are many types of genetically engineered mice: transgenic mice, knockin and knockout mice, and conditional...that only one chromosome needs to contain the transgene in order to see an effect. Hemizygous mice can...breed. Some problems with transgenic mice that can impact reproduction include embryonic lethality, a short...expression level, and site of integration of the transgene. These problems can be mitigated in different ...different ways. Prior to engineering a transgenic strain, the promoter being used should be tested in cell lines...

...StayGold variants AAV.CAP-Mac for systemic gene transfer in primate brains Endothelial-specific AAV delivery...electroporation or poor cellular entry with other transfection methods. The Junwei Shi Lab addressed this problem...variants:   Figure 2: Inner mitochondrial membranes imaged with structured illumination microscopy...2023). StayGold variants for molecular fusion and membrane-targeting applications. Nature Methods, 1–9. https...breaching the blood-brain barrier and delivering a transgene, with neuron bias observed in young macaques and... viral vectors for functional intravenous gene transfer throughout the non-human primate brain. Nature...the Gradinaru lab, AAV1-X1, was developed by transferring the 7-mer peptide region from variable region...

...AAAS Science and Technology Policy Fellow at the Veterans Health Administration. “But, seriously, what even...Science and Technology Policy Fellow (STPF) at the Veterans Health Administration, I could never get one straight...variety of backgrounds with technical expertise that ranges from astronomy to bioethics to chemical engineering...happen at the national level within the executive branch or in Congress, or can also happen at the state... early career researchers who have decided to transition out of academia from graduate school or a postdoc... came to DC to do either a sabbatical year or transition away from academia as tenured professors. There...taken through your own personal lens.  For me, the Veterans Health Administration was the perfect position...

...separation of the two DNA strands.  Annealing- The temperature is then dropped to a range in which the forward...the primers can anneal to the single stranded DNA template strands. DNA polymerase is also stable at this...template DNA to amplify from and unique single stranded DNA primers that bind upstream (5’) and downstream... process as DNA polymerases require an existing strand of DNA to add nucleotides to.  Using these reagents...reaction:  Denaturation- To amplify DNA, the two strands of the template DNA first have to be separated....used to quantitate RNA levels using reverse transcription-qPCR (RT-qPCR). The first step of this process...requires RNA to be converted to cDNA using reverse transcription and the cDNA is subsequently quantified by qPCR...

...vivo starts with transducing cells with the CRISPR library in vitro before transplanting them into mice....time. This reduces the chance of integration at random locations that could cause deleterious effects....portions of the AAV genome can integrate at double-stranded break points caused by CRISPR-Cas9 editing. The...The good news is that this integration is not random, nor is it a global occurrence, as integration sites...target cells or tissues. AAV also shows improved transduction efficiency over lentivirus in many mouse tissues...019-12449-2 Pupo, A., Fernández, A., Low, S. H., François, A., Suárez-Amarán, L., & Samulski, R. J. (2022..., U., Jabaudon, D., & Platt, R. J. (2023). Transcriptional linkage analysis with in vivo AAV-Perturb-seq...

...only have to worry about cloning your transfer plasmid. The transfer plasmids have different packaging capacities...AAV and lentivirus requires three plasmids — a transfer plasmid containing your gene of interest plus ...in mammalian cells is relatively quick. After transfection, HEK-293T cells start producing virus within...systems, it is standard to do only one passage (transfection). This differs from typical insect cell preparations...an additional couple of days, as it requires transduction of more cells, while AAV can be titrated following... baculovirus requires only two plasmids — your transfer plasmid and the baculovirus construct, called ...between insect and mammalian systems. Baculovirus transfer plasmids have a much larger packaging capacity...

...tool that responds strongly to magnetic fields (Frank Hayward et al., 2024). They deposited their plasmid...proteins. Motivated by curiosity Although a wide range of fluorescent proteins and optogenetic tools exist...when a small magnet was passed over the sample (Frank Hayward et al., 2024). The fluorescence intensity...the magnetic field is on. Image reproduced from Frank Hayward et al. 2024 under CC-BY license. They...mScarlet, and AsLOV2. After several rounds of semi-random mutagenesis and screening, the EGFP and mScarlet...the magnetic field is on. Image reproduced from Frank Hayward et al. 2024 under CC-BY license.   ...tools for biological applications across a wide range of topics. He embraces open science, self-publishing...

...or Förster] resonance energy transfer). FRET occurs when energy transfer from one FP (e.g. CFP) excites...classified by the emission color (emission wavelength range) FPs are usually classified by emission color as...as outlined below (or emission wavelenght range).  By mutating GFP, the variants blue FP (BFP), cyan FP...nm Green 499 - 519 nm Yellow 524 - 538 nm Orange 559 - 572 nm Red 574 - 610 nm Far-Red 625 -... ≥ 670 nm In addition to emission wavelength range, there are other traits that need to be considered... green or red light. For example, T-Sapphire, LSSmOrange, and LSSmKate. Fluorescent Sensors: These FPs...related colors. Furthermore, most FPs  have a broad range of emission which will be detected by longer-wavelength...

...Uniform Biological Material Transfer Agreement (UBMTA) for its material transfers. The UBMTA was drafted by...organizations Assurance that recipient may not use the material for a commercial purpose Assurance that recipient... has time for paperwork? Here’s why a Material Transfer Agreement (MTA) matters: Science thrives on collaboration...universities and institutions require them for material transfers. When Addgene entered the plasmid sharing space...receiving institution. Therefore, many technology transfer officers are familiar with the UBMTA and are happy...  material transfer agreement...

...dCas9 has focused on transcriptional activation using a fusion to a transcriptional activator such as VP64...repression of transcription through binding a promoter region to inhibit association of transcriptional activators...locus of interest, the promoter region of the transcription factor IRF-1. Immunoprecipitation, using an ...2018). Furthermore, the Fujii Lab generated a transgenic mouse line and retroviral expression system for...Tapscott Lab identified regulators of DUX4, a transcriptional regulator that when inappropriately expressed...PMCID: PMC6001023. Fujita, Toshitsugu, et al. "Transgenic mouse lines expressing the 3x FLAG‐dC as9 protein...CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes." Cell 154.2 (2013): 442-451. ...

...false positive colonies. Transformation - the linear fragments are transformed into E. coli and plasmid...assembly of a circular plasmid in vitro, before transforming it into E. coli for propagation. However, while...using a simple two-step, 2 hr protocol prior to transformation (Figure 1). As this approach requires no special... encode homology arms to allow immediate co-transformation with linearized vector backbone. DpnI digestion...failed so far. What is most important is the transformation efficiency of the strain: when asking the cell...high fidelity DNA polymerase This will minimize random errors during PCR: we recommend Phusion or Q5. ...investigating the molecular biology of synaptic transmission and plasticity.     Javier García-Nafría is ...

... dilution should fall within the range of the assay for a wide range of titers. We usually dilute our ...determine if your titers are within the expected range. We have also found that generating the standard...For these reasons, we are in the process of transitioning our titering method from qPCR to ddPCR. Droplet...virus. It is important to note that the dynamic range of the ddPCR is between 1 and 100,000 genome copies...per reaction. Since AAV titers tend to be in the range of 1012 to 1013 GC/mL, you must serially dilute ...million.  After making the dilutions, they are transferred to a new plate containing the mastermix which...generator, you will have to be careful about transferring your droplets from the droplet generator to ...

... inducible translocation trap (ITT) system to analyze signal-induced nuclear translocation of proteins...functions such as epigenetic regulation and transcription by using locus-specific chromatin immunoprecipitation...biochemical analysis of genome functions such as transcription and epigenetic regulation. To elucidate molecular...Addgene realizes the One-Stop shopping for a wide range of plasmids. The merit of depositing plasmids to...considering the peer-review system of journals and grant applications, for example. However, at the same ...living for Japanese, which is not trivial, and the grant situation is currently much better than that in ...tough competition for academic positions and poor grant situation. So, young researchers need to publish...

... of fluorescence: For applications that involve ranges of pH, and significant lengths of time, the stability...receptor conformation and signaling activity​​ (Sridharan et al., 2014). It's also crucial to evaluate how...the background. Dyes that can pass through cell membranes can prove useful for live-cell imaging. Experimental...considerations regarding the choice of fluorescent dyes (Sridharan et al., 2014). Manufacturers will typically recommend...accurate tagging. In Fluorescence Resonance Energy Transfer (FRET) applications, the dyes need to have compatible...and emission profiles to enable effective energy transfer, which is key to observing interactions and conformational... specifically to the AT-rich regions of double-stranded DNA and exhibits a blue-fluorescent light when...

...injections can be found here [5, 6]. Opsin transgenic mice Transgenic mouse lines have also been engineered...specific brain region can result in a particular neurotransmitter release or post-synaptic current. Fortunately...available, instructions for obtaining a material transfer agreement, and list of vendors (such as the vector...at a similar spectrum as HR and also results in membrane hyperpolarization but unlike HR, this is achieved...[10]. Unfortunately, opsins are not actively transported and must passively diffuse so, depending on the...axonal tracing with fluorescent proteins in nontransgenic and cre driver mice. Current protocols in neuroscience...et al. A toolbox of Cre-dependent optogenetic transgenic mice for light-induced activation and silencing...

...protease allows a transcription factor to enter the nucleus and modulate transcription of the mCitrine ...HeLa cells were transfected with plasmids encoding FPs tagged with the plasma membrane targetin sequence...non-specific labeling. For labeling the plasma membrane, they tested different target sequences for improved...expression can vary significantly depending on transgene design and delivery. To overcome these variations...into genomic DNA with the help of the piggyBa transposase. Image from Hazelbaker et al., 2020. The lab...activating expression of TCF4 while monitoring transcript and protein levels. Plasmids for multiplexed ... new serotype PHP.V1 that exhibits efficient transduction of vesicular brain cells. Find this AAV at ...

...amplification) or reverse transcriptase (RT)-RPA, respectively. RPA is coupled with T7 transcription to convert amplified... method termed DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR) (Chen et al., 2018). Both SHERLOCK...almost anywhere as lateral flow strips are easy to transport and work rapidly, providing reliable results in...    DETECTR- DNA Endonuclease Targeted CRISPR Trans Reporter Cas12a (Cpf1) is a CRISPR Cas variant that...discovered Cas12a’s ability to cleave non-specific (trans) ssDNA and harnessed this ability to create a DNA...DNase activity of Cas12a is initiated. Surrounding trans-ssDNA, including the ssDNA-FQ reporter are subsequently...Servellita V, Singh J, Miao X, Streithorst JA, Granados A, Sotomayor-Gonzalez A, Zorn K, Gopez A, Hsu ...

...promoter of another gene, thereby driving aberrant transcription. That being said, we generally pick guides...been significant work on constructing Cas9 transcriptional activators. These constructs allow for the ...Cas9 deficient in DNA cutting activity to a transcriptional activation domain (Fig 1). Two ways to use...picking whichever guides are closest to the transcription start site (TSS). We recommend targeting the...Latency Reversal Using CRISPR/Cas9-Derived Transcriptional Activator Systems. PLoS ONE 11:e0158294 . https...Church GM (2015) Highly efficient Cas9-mediated transcriptional programming. Nat Methods 12:326–328 . https...Nishimasu H, Nureki O, Zhang F (2014) Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex...

... cell types. Specific transgene expression can be also achieved in nontransgenic animals by using cis ...delivery by increasing transgene cargo capacity, enhancing anterograde transport efficiency, and engineering...proteins that determine the AAV serotype. During transduction, the viral capsid proteins bind to the target...serotype used for a specific cell type. Find some transduction characteristics of select AAV serotypes in Table... AAV vectors are common approaches to control transgene expression by restricting expression to genetically...enhancers, multicistronic vectors, and post-translational regulatory elements. Examples of these approaches...

...enzyme or transcriptional activation, conformational changes, translation, and protein translocation. Targeting...triggers CaM/M13 interaction that increases FRET transfer between the fluorescent proteins. FRET is highly...characteristics like pH, redox state, and voltage across membranes. These sensors generally follow the same pattern...an organelle-specific targeting signal or a transmembrane domain, biosensors can be targeted to a particular...endoplasmic reticulum (ER), vacuole or cellular membranes. Examples include blue flame mitochondria-targeted...