...identification.   Fig. 2: Gel image of Plasmid 61564: pBAMD1-2 (~4.7 kb) digested with SpeI and...to the formation of plasmid multimers. Cell. 1976 Dec;9(4 PT 2):707-16. doi: 10.1016/0092-8674(76)90134... at ~3.6 kb and ~1.1 kb (lane 3). Uncut DNA (lane 2) has a band at <3 kb, which corresponds to the supercoiled...dimers in Escherichia coli K-12. Plasmid. 1989 Mar;21(2):138-41. doi: 10.1016/0147-619x(89)90057-7. PMID: ...Plasmids and Chromosomes. Microbiol Spectr. 2014 Oct;2(5). doi: 10.1128/microbiolspec.PLAS-0025-2014. PMID...Behavior of Plasmid DNA. Microbiol Spectr. 2015 Apr;3(2):10.1128/microbiolspec.PLAS-0036-2014. doi: 10.1128...at lower copy numbers within bacterial cells, and bacterial cells with multimers grow more slowly compared...

...low expression, and everything in between (Figure 2). These smears tend to occur when you are looking ...on your experiment. You can see from Figure 2 that determining where to place your gate based on the dot... dot plot would be complicated.  Figure 2: (A) a smeary dot plot showing a range of antibody readouts...plot in Figure 4 compared to the dot plot in Figure 2. Quantification using MFI makes it easier to identify...percentage of total live cells are GFP+. In this plot, with adjusted voltage, cells that did not receive ...would serve you well. Percentage of cells in a parent gate (all live cells for example) that are positive ...example, you have introduced a GFP protein into your cells (visualized via the FITC channel) and you are trying...

... we identified in step 2. The result is a heterogenous population of stem cells, usually housed in a single...: 1. Find Enhancers that have NANOG binding sites 2. Design a CRISPR screen to target and disrupt the ...NANOG enhancers 3. CRISPR out the enhancers from step 2. See which ESCs die or differentiate Step 3 reveals...expression for NANOG enhancers, we’re ready for step 2: design a CRISPR screen to disrupt these enhancers...gRNA sequences without off-target effects for the 2+ million DHS sites. See below for further details.... for stem cells, or metastasized tumor cells, or alzheimers-affected neurons, or [your cell line of interest...critical to stem cell survival. Knowing which genes influence the survival of our cell culture is the foundation...

...question: C2c1, C2c2 and C2c3 (short for Class 2, candidate 1, 2, or 3). C2c1 and C2c3 are related to Cpf1,...characterization of diverse class 2 CRISPR-Cas systems." Molecular cell 60.3 (2015): 385-397. PubMed PMID...however cutting efficiency of Cas13a is reduced when 2 mismatches are present. The protospacer flanking sequence...PMC5127784. Find plasmids from this paper at Addgene. 2. East-Seletsky, Alexandra, et al. "Two distinct RNase...thought to activate programmed cell death or a dormant state for bacterial cells that have been infected with...bacterial cells, LwaCas13a does not display nonspecific RNA cleavage activity in eukaryotic cells. They arrived...target RNAs, although its usefulness in bacterial cells will likely be limited by the propensity of Cas13a...

...Tethering partner Subcellular localization 1 TagBFP 399nm/ 456nm histone 2B (H2B) Chromatin 2 Cerulean 433nm...based (chaining-based) cloning. The modules (Figure 2) are contained in individual plasmids and are each...between various cloning methods are listed in Table 2. For example,  chaining-based cloning methods require...suitable for combining endogenous sequences. Table 2: High Throughput Cloning Strategies Method Technology...PMID: 20205762. PubMed Central PMCID: PMC2822740. 2. Engler C, Kandzia R, Marillonnet S. A one pot, one...protein localization, as well as cellular processes like gene expression and cell migration (Sladitschek and... MXS-chaining were used to label subcellular structures in live cells. Four individual expression constructs...

...PubMed Central PMCID: PMC3640491. 2. Grimm, D., et al. "Titration of AAV-2 particles via a novel capsid ELISA...report the physical titer of a viral solution (Figure 2). Because physical titers are used for dosing purposes...mean and how they can be compared.   Figure 2: Label that comes on Addgene’s AAV aliquots. Our titer...of genomes can limit production of recombinant AAV-2." Gene therapy 6.7 (1999). PubMed PMID: 10455443. ...Characterization of a recombinant adeno-associated virus type 2 Reference Standard Material." Human gene therapy 21.10...particles that can transduce cells. Infectious titers are typically quantified by cell transduction assays. Wild-type...

...Citorik Plasmids here). In both cases, they used 1-2 CRISPR crRNAs targeting sequences found only in certain...resensitize antibiotic-resistant bacteria (Figure 2). The first phage introduced is lysogenic, integrating...machinery targeting 1) the given resistance gene and 2) a second lytic phage. Bacteria that receive this ...bacteria have previously flourished.   Figure 2: Two-phage strategies to resensitize bacteria to antibiotics...237163. Find plasmids from this paper at Addgene. 2. Bikard, David, et al. “Exploiting CRISPR-Cas nucleases...reach essentially every cell or else small pools of antibiotic resistant cells could repopulate the environment...strategies are nonspecific - they harm any bacterial cell without a resistance gene, allowing resistant bacteria...

...kinetics which are illustrated in figures 2 and 3.  Figure 2: Glutamate titration curves for the different...signals upon sensing glutamate using both 1-photon and 2-photon imaging.   Figure 1: 1P and 2P excitation...adapted from Aggarwal et al…, Supplementary Figure 2.   Improved trafficking and localization through... glutamate release from neurons and other brain cells in vivo. Glutamate plays a variety of roles in synaptic...sensor to the membrane and displays it on the extracellular side. In addition to the original PDGFR TMD,...promoter, FLEXed for expression in Cre-expressing cells hSynap: Human Synapsin-1 promoter. Good for neuronal...promoter, FLEXed for expression in Cre-expressing cells. hSynap-FLP: Synapsin-1 promoter, FLP-dependent ...

...Currently available from the Kim lab are 2 presynaptic and 2 postsynaptic targeting mGRASP plasmids. Additionally... Robinson et al., Dev Cell 2010 Feb 16;18(2):324-31. Cheeseman et al., J Cell Sci 2013 May 1;126(Pt ... 2µg/ml of Doxycycline for 2 hours, and imaged on the Keyence BZ-9000 fluorescence microscope 8 hours ...promotes the formation of an isopeptide bond between 2 peptide tags, SpyTag and KTag. The group demonstrated... of three sets of cloning vectors (Level 0, 1, or 2) which can be utilized in three successive assembly...1 modules can be similarly assembled into a Level 2 vector, thus forming a functional genetic circuit....circuit. Flexibility has been built into the Level 2 vectors to allow for additional iterations of Level 1 assembly...

...purify IVT sgRNA in the lab. Figure 2: Cas9 RNP complexed with 2-part cr:tracrRNA tags genes more effectively...is then transfected into stem cells. Cells are allowed to recover for 2-5 days and then analyzed for tagging...transfect and let cells recover for 2-5 days.Note: We use the Nucleofection method for delivery of csRNP... knock-in efficiency (<2% positive by ICC for the tag) or do not see any cells positive for tag insertion...stem cells. Development 144: 635-648. PubMed PMID: 28096221. PubMed Central PMCID: PMC5312033. 2. Dewari...IVT sgRNA) approach (Dewari et al. 2018 and Figure 2). Not only is the cr:tracrRNA approach highly efficient...transfected cells, we count the number of tag-positive cells (tag ICC) as a percentage of total cells in the...

...either SARS-CoV-2, Adenovirus type 41 (AdV-41), or Adenovirus-Associated Virus type 2 (AAV2) – or some... was either 1) a physiological effect of SARS-CoV-2 toward destabilizing one’s natural immune response...response to Adenovirus (and/or to its partner, AAV) or 2) a sociological effect of the COVID-19 lockdowns causing...… Chiu, C. Y. (2023). Adeno-associated virus type 2 in US children with acute severe hepatitis. Nature... AdVs is dependent on modulation of the host cell’s cell cycle; inhibition of the innate immune system... in the producer cells where there is E1 and E3 gene complementation. In target cells that are infected...hexon), as well as other intracellularly-exposed proteins and downstream cellular events, all recruit strong...

...plasmid, or a 2-plasmid system in which Cas9 must be delivered separately.  Mutant cells are enriched ...targeting a specific gene within the genome (see Fig. 2). Individual gRNAs are designed in silico using publicly...mouse, human, fly, E. coli, and T. gondii genes.  2) What genetic modification are you trying to make?...CRISPR libraries is a multi-step process (see Fig. 2). In most cases, CRISPR libraries are provided at ...heterogeneous population of mutant cells, with each cell or set of cells containing a mutation in a different...fluorescence-based cell sorting and screened for a particular phenotype. For example, mutant cells can be used...lead to skewed results. Selecting a cell type - Theoretically, any cell type can be used in a CRISPR screen...

..., AAV2/8 has a type 2 ITR and a type 8 serotype. Almost all vectors contain type 2 ITRs, which comes from...terminally differentiated, non-dividing cells.   Figure 2: DNA rescue and transduction of a conventional...quantitative PCR-based method for the titration of AAV-2 vector stocks."Molecular Therapy 6.2 (2002): 272. ...Time to expression AAVs have to infect cells, hijack host cell machinery, and synthesize a second strand...that can be used to express genes of interest in cell and animal models. AAVs are non-pathogenic, do not...be specifically targeted to a number of different cell types even when systemically administered in an ...ranging from transient gene expression in a particular cell type to CRISPR genome editing, optogenetics, and...

... 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...can be used in bacterial and cell-free expression systems, mammalian cells, yeast, flies, and even plants...

...place. Then plasmids are cured from the cell. Steps 1-1 and 1-2 can be done simultaneously. Image from ...1) replaced the promoter of gRNA-pMB1 with PrhaB, 2) changed the replicon of pCas into a non-temperature-sensitive... pEcCas/pEcgRNA system in 4 E. coli K-12 strains, 2 E. coli B strains, 1 E. coli W strain, and 1 Tatumella...MUCICAT better, it is faster. Using MUCICAT only needs 2 newly constructed plasmids (pDonor and pQCascade) ...rounds of transformation in 30 days. Figure 2: (A) MUCICAT plasmids for multicopy integration. (...construct and then introduce both plasmids into the cell. The edit in the chromosome can now take place. .... After editing, pTargetF can be cured from the cell by inducing expression of the sgRNA that targets ...

...//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 ...Syndrome and Related Disorders. Cell 152:984–996 . https://doi.org/10.1016/j.cell.2013.01.038 Guy J, Gan J, ...Programmable RNA Editing. Cell Reports 32:107878 . https://doi.org/10.1016/j.celrep.2020.107878 Sinnett SE...deliver the precise amount of protein to each brain cell to restore physiological levels. Much research is...

...testing your AAV.  Figure 2: Comparison of AAV serotypes 1, 2, 5, 6, 8, and 9 in two different ...your experimental conditions and measurements; (2) the cells you’re targeting; (3) how much expression is... Tropism and Axonal Transport of AAV Serotypes 1, 2, 5, 6, 8 and 9 in the Mouse Brain. PLOS ONE, 8(9),... Comparison of four promoters. Gene Therapy, 28(1–2), 56–74. https://doi.org/10.1038/s41434-020-0169-1...analyses of adeno-associated viral vector serotypes 1, 2, 5, 8 and 9 in marmoset, mouse and macaque cerebral... where many different cell types, or even different phenotypes of the same cell type, are intermingled...postsynaptic cells when used at very high titers (Zingg et al., 2017), which can lead to cells in far away...

... recombination at the cellular level in the mouse. 2003 PubMed PMID: 12665802. 2. Branda, CS and Dymecki...2008. A FLEX switch targets Channelrhodopsin-2 to multiple cell types for imaging and long-range circuit ...conditional knock-in cassette. As shown in Figure 2, the cassette should contain a reporter DNA sequence...a targeted neuronal population.    Figure 2 (modified from Schnutgen et al. Nat Biotech 2003):...delivered using AAV injection, the majority of the cells surrounding the injection site will become infected... ensure that the reporter remains silent until a cell or tissue-specific Cre is provided. Since the FLEx...used to study the physiology or behavior of certain cell populations.  Acknowledgement:Through a partnership...

...for retrograde transport has been found for AAV1, -2, -5, -7, -8 (Taymans et al., 2007), AAV1 (Hollis et... tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. PLoS One 8:e76310...reveal novel hippocampal CA2 circuits. Nat Neurosci 17(2), 269–279. PubMed PMID: 24336151. PubMed Central PMCID... Mechanisms of Motility and Regulation. Neuron 84(2), 292–309. PubMed PMID: 25374356. PubMed Central PMCID...transduction efficiency of recombinant AAV serotypes 1, 2, 5 and 8 in the rat nigrostriatal system. J Neurochem...analysis of adeno-associated viral vector serotypes 1, 2, 5, 7 and 8 in mouse brain. Hum Gene Ther 18, 195–...retrograde access to projection neurons. Neuron 92(2), 372–382. PubMed PMID: 27720486. PubMed Central PMCID...

...of conventional gene targeting is lowered to about 2-3 months. But certain models remain more difficult... 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...Cas-mediated genome engineering." Cell 154 (2013):1370-9. DOI: 10.1016/j.cell.2013.08.022 PubMed: PMID: 23992847...then electroporate it into mouse embryonic stem cells. After selecting for clones with the recombined ...