...one of lox2272 sites will now be sandwiched between 2 loxp sites and is excised by Cre (Figure 1). This ...flank the transgenes (non-homologous pairs) (Figure 2.). They found that almost all off-target expression...exercise caution when using them.    Figure 2. Schematic diagram showing the mechanism of spontaneous... Tropism and Axonal Transport of AAV Serotypes 1, 2, 5, 6, 8 and 9 in the Mouse Brain. PLoS ONE 8:e76310... Pseudotyped with Viral Capsids from Serotypes 1, 2, and 5 Display Differential Efficiency and Cell Tropism... with the same titer and serotype previously used in a different brain region that the same result will...sites will be flanked by two lox sites now in the same orientation. Next, the loxp site is excised since...

... Figure 2: Due to differences in their size and complexity, cells from a whole blood sample will separate...termed fluorescence activated cell sorting or FACS (2). By analyzing single cells instead of the population...expression. A routine flow cytometry experiment uses 2 or 3 different fluorescent colors each measuring a...distinct populations on a FSC versus SSC plot (Figure 2).  A user can then “gate” around the specific population...cell and T cell into distinct populations (Figure 2). An immunologist who studies B cells, might then ...was raised in the same host species, of the same Ig subclass, and conjugated to the same fluorochrome as...ensure that samples are single cell suspensions. For cells that tend to aggregate, pass the sample through...

...for controlling neuronal activity, channelrhodopsin-2 (ChR2), a blue light-activated cation channel, from...used in tandem with the opsin.      Figure 2: Diagram of excitatory (ChR2) activation by light ...) domains, phytochrome B (PhyB), and cryptochrome 2 (CRY2) (Zhang & Cui, 2015). When stimulated with light...comparative analysis of microbial opsins. Nature Methods, 9(2), 159–172. https://doi.org/10.1038/nmeth.1808 References...comparative analysis of microbial opsins. Nature Methods, 9(2), 159–172. https://doi.org/10.1038/nmeth.1808 Nagel...Hegemann, P., & Bamberg, E. (2003). Channelrhodopsin-2, a directly light-gated cation-selective membrane ...intracellular signaling pathways. Trends in Biotechnology, 33(2), 92–100. https://doi.org/10.1016/j.tibtech.2014.11.007...

...discussed them in meetings and 2) creating a pictorial guide for our engineers (Fig. 2). Creating this guide was...communication has helped Addgene move from completing 2-3 large projects a year, on average, to completing... and engineers were attending the same meetings and hearing the same words, they were leaving these meetings...aforementioned lab manager, said the word “sample,” did the word convey the same concept to Morgan, our senior engineer...help get everyone on the same page. Others in the meeting might also have the same question and asking can...When everyone at a meeting is using the same words with the same meanings consistently, meetings will be...counsel, used the word “service,” did he mean it the same way as Bryan, our customer support manager? When...

...month, from 1/22/20–2/23/20, where there were zero plasmid requests. Afterwards, from 2/24/20–3/15/20 (the...Each paper has between 1–32 plasmids and a total of 2–2,590 requests. We received plasmid requests from ...observed between Jan. 1 and Mar. 1, 2020. Figure 2: A simple approximation of the effect of COVID-19 ...However, it is evident that if we believe that 1/22/20–2/23/20 are the more accurate dates of lockdown in China...interpretation -- the effect would generally not be the same if weekends were somehow imposed on scientists. ...

...incubation at 37 °C. Wash the membrane for 5–10 minutes 2–3x in fresh wash buffer such as PBS or TBST. Stringent...Purpose Tris HCl 62.5 mM Buffer SDS 2% (w/v) Detergent Β-mercaptoethanol (βME) 100...minutes at 50 °C. Wash the membrane for 5–10 minutes 2–6x in fresh wash buffer such as PBS or TBST. Note ...membrane will remove some of your sample —and harsher stripping means more sample loss. If you are still optimizing...you want to detect more than one protein in your sample? Or what if something weird happened during your..., almost like new. This approach preserves your sample and saves you from having to run a whole new gel...stripping solution (see below) to minimize loss of your sample from the membrane. Next, plan out the order of ...

...FLP and Cre for additional control   Figure 2: Expression of a morphological marker (eYFP) and synaptic...Sternson. 2008. “A FLEX Switch Targets Channelrhodopsin-2 to Multiple Cell Types for Imaging and Long-Range ...PMID: 18614669. PubMed Central PMCID: PMC2593125. 2. Buchholz, F, P O Angrand, and A. Francis Stewart....The FLP Recombinase of the Saccharomyces Cerevisiae 2 Micron Plasmid Attaches Covalently to DNA via a Phosphotyrosyl...reintroduction, inversion and re-inversion happen at the same probability. Continuous inversion of the DNA sequence...recombination can only occur between target sites of the same sequence in FLEx vectors, the DNA sequence that ...neuron and its synapse should be visualized at the same time from two different plasmids this approach becomes...

...imaging (Figure 2). If you’re working with purified proteins in vitro, you can even use the same tag for multiple...super-resolution or single-molecule techniques.   Figure 2: Two-color super-resolution imaging of ER and mitochondria...stimulated emission depletion (STED) nanoscopy. Scale bar, 2 μm. Inset shows comparison with standard confocal ...Labeling in Living Cells. Chemistry & Biology, 15(2), 128–136. https://doi.org/10.1016/j.chembiol.2008.01.007...conjugates: A flexible chemical tag. Nature Methods, 2(4), Article 4. https://doi.org/10.1038/nmeth749 Strebinger...sequentially adding different ligands to the same sample to label sub-populations of your protein of interest...probably find lots of different plasmids containing the same protein of interest tagged with different fusion...

...therapy had fallen - plummeted really -  from grace 2 years prior with the death of Jesse Gelsinger in an... the whole field to a grinding stop and only now, 2 decades later, is it finally back in the limelight...the other hand, have broad tropism in the eye. AAV-2, the first identified serotype and the one used in...reading 1. FDA approves gene therapy for blindness 2. Gene Therapy emerges from disgrace to be the next...retinal cells - AAV - were becoming available. At the same time advances in human genetics led to the identification...24664759. PubMed Central PMCID: PMC4267879. 10. Samulski RJ, Muzyczka N., 2014. AAV-Mediated Gene Therapy...

...is excised from the donor vector.   Figure 2: The Gateway system adopts phage integration into ...our website to search for your favorite gene! STEP 2: Generate an Expression Clone When making the expression...expression construct in only 1 day, as opposed to 2+ days with traditional restriction and ligation cloning...Clon Transgen 4:138. doi:10.4172/2168-9849.1000138 2. Hartley JL. Use of the Gateway System for Protein...possible to set up the BP and LR reactions in the same tube, speeding up the cloning of the attB-PCR products...multiple DNA fragments into many vectors at once in the same tube. You can clone up to 4 DNA fragments, in a ...

...plasmid-based expression requires serial transfection of 1 or 2 plasmids that express the reprogramming factors of...for generating iPSCs by plasmid transfection where 2 of 11 clones tested had plasmid integration. Another...understanding the mechanisms of reprogramming, and 2) studies with clinical endpoints. In the first scenario...PubMed PMID: 27764162. PubMed Central PMCID: 5072705. 2. Sommer, Cesar A., and Gustavo Mostoslavsky. "The ...challenges." Journal of Cellular Physiology 228, no. 2 (2012): 267-75. PubMed PMID: 22767332.  3. Kim, Dohoon...PubMed PMID: 23546745. PubMed Central PMCID: 4176696. 2. Yusa, Kosuke, Roland Rad, Junji Takeda, and Allan...generation. Expert opinion on biological therapy, 10 2, 231-42. PubMed PMID: 20088717. PubMed Central PMCID...

...of conventional gene targeting is lowered to about 2-3 months. But certain models remain more difficult...included in the final ssDNA template. Figure 2: Overview of ivTRT to generate an ssDNA repair template... floxed conditional allele requires you to target 2 loxP sites to intronic regions 0.5 - 0.8 kb apart....Typically, Easi-CRISPR works best with insertions of 2 kb or less. Luckily, most of the commonly used cassettes... induction systems (ttA or rtTA) are between 1 to 2 kb in length. There are plenty of Addgene plasmid ...create the ssDNA construct as described in Figure 2.  You can also use Addgene plasmids to purify Cas9...They have successfully used guides targeting the same or complementary strands, as well as guides facing...

...experimental samples. Let's look at an experimental example in Figure 2. Figure 2A shows the sample prior to... Figure 2: Compensation example based on experimental sample. Flow plots showing a sample before (A)...in Figure 2, following compensation. Figure 3: Flow plot for a double positive sample. The cell... your experimental samples In order to do compensation on your experimental samples, you will first need...apply to your experimental samples as they did with your single control samples: if the population needs...fluorescent channel to another. When you label your samples with multiple antibodies, the fluorescent probes...general, if two fluorescent markers are read by the same laser, they will need to be compensated for one ...

...//doi.org/10.1002/1096-8628(200022)97:2<147::aid-ajmg6>3.0.co;2-o Baker SA, Chen L, Wilkins AD, Yu P, ...Caused by a mutation in the methyl-CpG binding protein 2 (MECP2) gene, Rett Syndrome is a rare, progressive...X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat Genet 23:185–188 . https://doi.org/10.1038/13810... (2000) Rett syndrome: Methyl-CpG-binding protein 2 mutations and phenotype-genotype correlations. Am ...https://doi.org/10.3389/fgene.2019.00625 McGraw CM, Samaco RC, Zoghbi HY (2011) Adult Neural Function Requires...

...   Figure 2: Synthetic promoters can be paired for targeted expression. This same pairing of synthetic...retinal cell-type specific genes from mice. Strategy #2 (ProB): Create promoters using phylogenetically conserved...1345-1356. https://doi.org/10.1038/s41593-019-0431-2  Neff, E.P. An AAV library for retinal cell expression...different synthetic promoter driving expression of the same transgene: an optogenetic reporter, channelrhodopsin...in mice is not a good predictor for targeting the same cell class in NHPs (R=0.38) and humans (R=0.32),...), but the odds of that promoter targeting the same cell class in all three species are still greater ...in monkeys was a good predictor for targeting the same cell class in humans (R=0.67).  A database of the...

...phosphorylation events in the proteomes of higher mammals (2). How can we begin to understand the importance of... the open reading frame (ORF) of interest (Figure 2). 1. The phosphoserine OTS The backbone of the phosphoserine...optimized Sep-OTSλ and can recommend its use (6).  2. C321, a specialized bacterial host strain for pSer-incorporation...biology4.5 (2002): E127-E130. PubMed PMID: 11988757. 2. Hornbeck, Peter V., et al. "PhosphoSitePlus, 2014... purified from the system, researchers can run samples on SDS-PAGE gels containing Phos-Tag, a small molecule...non-phosphorylated serine at the TAG codon of the same expression vector used for phosphoserine incorporation...incorporation of serine) population of protein using the same expression plasmid. This is a particularly important...

...design primers to detect the presence of your insert; 2) set up a standard PCR reaction (primers, dNTPs, polymerase...strategies for primer design: 1) insert-specific primers, 2) backbone-specific primers, and 3) orientation-specific...negative clone (-) will not. Backbone-specific primers (2) give larger sized products for positive clones (+...briefly boiling the sample before use or by directly adding a small volume of the sample to the PCR reaction...enzyme digest; however colony PCR can accomplish the same thing in less time and for less money. The key steps...also great for screening clones created with the same backbone but that contain different inserts. When...sequence of the insert is, allowing you to use the same primer pair to screen for the presence of many different...

...purify IVT sgRNA in the lab. Figure 2: Cas9 RNP complexed with 2-part cr:tracrRNA tags genes more effectively...into stem cells. Cells are allowed to recover for 2-5 days and then analyzed for tagging efficiency using...IVT sgRNA) approach (Dewari et al. 2018 and Figure 2). Not only is the cr:tracrRNA approach highly efficient...cell suspension, transfect and let cells recover for 2-5 days.Note: We use the Nucleofection method for delivery...success was ~%50. If you get low knock-in efficiency (<2% positive by ICC for the tag) or do not see any cells...PMID: 28096221. PubMed Central PMCID: PMC5312033. 2. Dewari PS, Southgate B, Mccarten K, Monogarov G, ...turnaround time 5-7 days); because the tracrRNA is the same in all csRNP experiments, it can be ordered in bulk...

... Multiple Introns.” Plant Communications, vol. 2, no. 2, Elsevier BV, Mar. 2021, p. 100135. Crossref, ...Constructs.” PLoS ONE, edited by Jean Peccoud, vol. 6, no. 2, Public Library of Science (PLoS), 18 Feb. 2011, p... vol. 5, no. 10, American Chemical Society (ACS), 2 May 2016, pp. 1059–69. Crossref, https://doi.org/10.1021...of Plant Genomes.” The Plant Genome, vol. 16, no. 2, Wiley, 9 Mar. 2023. Crossref, https://doi.org/10.1002...Crossref, https://doi.org/10.1186/s12870-020-02388-2. Stuttmann, Johannes, et al. “Highly Efficient Multiplex...Crossref, https://doi.org/10.1038/s41598-019-46171-2. Resources on Addgene.org Modular Cloning (MoClo...unique genomic targets or multiple sgRNAs against the same target to ensure efficient cutting. Beyond sgRNAs...

...transcription factors 1 and 2. See Aranda-Díaz et al. for more information. The El-Samad lab predicts that this...difficult-to-manipulate primary cells, achieving knockdown within 1-2 hr of reagent delivery. TRIM-Away is suitable for ...factor 1 controls expression of transcription factor 2, which directly controls expression of a reporter ...Factors Podcast Segment Researchers from the El-Samad lab recently deposited plasmids encoding synthetic...encoding these synthetic transcription factors, the El-Samad lab has deposited plasmids encoding fluorescent ...