...them in Regenerative Medicine. Acta Naturae, 2010. 2(2): p. 18-28. PubMed PMID: 22649638. PubMed Central...detail below [2]. Multipotency: When a cell can differentiate into multiple cell types of the same lineage....capability to differentiate into specialized cell types [2]. We general speak of 4 levels of stem cell potency...zygote through to the morula stage are totipotent [2-4]. Pluripotency: When a cell has the ability to differentiate...PMID: 26726264. PubMed Central PMCID: PMC4686367. 2. Jaenisch, R. and R. Young, Stem cells, the molecular...

...modules (Figure 2) are contained in individual plasmids and are each flanked by the same multiple cloning... TagBFP 399nm/ 456nm histone 2B (H2B) Chromatin 2 Cerulean 433nm/ 475nm Lyn-tag (derived from the tyrosine-protein...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...e.g., a set of various mammalian promoters), the same cloning procedure can be used to incorporate each...

.... Neuron 84(2), 292–309. PubMed PMID: 25374356. PubMed Central PMCID: PMC4269290. Masamizu, Y., Okada,...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...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...

...typically, normal (bell curve) or skewed (see Fig. 2). Assumptions – characteristics of a data set; ...levels of variance among data.     Fig. 2: Examples of data distribution profiles.   Parametric... positive is much higher than if you only look at 2 or 3 pairwise comparisons. Similar to family-wise ...statistical facets of reproducibility. Adv Physiol Educ. 40(2): 248-252 (2016). 0.1152/advan.00042.2016 Finch, W...Simulation Study. Front. Appl. Math. Stat. Volume 2 (2016). doi.org/10.3389/fams.2016.00002  ...comparisons. Paired and unpaired t-tests Samples that come from the same subject or population - before and ...referred to as two-sample or independent, but these designations all refer to the same test. On some occasions...

...Golden Gate Assembly is commonly used to assemble 2–10 DNA fragments in a single “one-pot” reaction to...the transformants onto the selection plate. Figure 2 shows representative transformation plates obtained...fidelity studies conducted by Potapov et al. at NEB (1, 2). While T4 DNA Ligase, the mainstay of most biotechnological... (percent correct) Calculated colony totals Per 2 μl assembly reaction Per full assembly reaction** ...calculated yields from entire outgrowth built from 2 µl of the assembly reaction, and from the entire assembly...fidelity and bias profiling." bioRxiv (2018): 322297. 2. Potapov, Vladimir, et al. "A single-molecule sequencing...confirmed the insertion of the Lambda insert at the same high frequencies as the antibiotic selection markers...

...dCas9 and an eGFP tag, and 2) gRNAs Number of loci targeted One Many Sample type(s) Cell lines Cell ...Originally published Dec 7, 2017 and updated Jul 2, 2020. Promoters may be the star of gene regulation...sequence targeted in the experiment. Figure 2: Comparing CAPTURE and CAPTURE 2.0.     Original...cell line expressing 1) FLAG-biotin-tagged dCas9, 2) BirA, and 3) one or more gRNAs Two separate lentiviruses...identified and the level of on-target enrichment. 2. Determining the spatial and hierarchical organization...Cytogenetics, 11. https://doi.org/10.1186/s13039-018-0368-2  Liu X, Zhang Y, Chen Y, Li M, Zhou F, Li K, Cao H...

...genome (Figure 2). While it is technically possible to encode all four viral genes in the same plasmid (as...study aspects of the EBOV replication cycle under BSL-2 conditions (4). What is a minigenome?   The EBOV ...transcription by the EBOV polymerase (reviewed in 2, 5). The minigenome is capable of being replicated...nonsegmented negative sense RNA viruses (reviewed in 2, 3). Other important components of the Zaire ebola...500 ng NP, 500 ng VP35, 100 ng VP30, 100 ng L, and 2 μg minigenome plasmid per well (in a 6 well plate ...PubMed PMID: 9847328. PubMed Central PMCID: PMC103829. 2. Conzelmann, K. K. "Reverse genetics of mononegavirales...

...Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Figure from Zetsche et al., 2015...2015. How was Cpf1 found and tested? Class 2 CRISPR systems, including the type II Cas9-based system...employed by class 1 systems. A putative new class 2 nuclease, Cpf1 (CRISPR from Prevotella and Francisella... the 3’ end of the target sequence. Figure 2: Effects of (A) double and (B) single base pair gRNA-target...Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system." Cell (2015). PubMed PMID: 26422227... Find plasmids from this publication at Addgene. 2. Zetsche, Bernd, et al. "Multiplex Gene Editing by...cleavage are 42-44 nucleotides in length, about the same size as Cas9’s, but with the direct repeat preceding...

...use 2 different secondaries for your antibody pair and visualize both targets in parallel (Fig. 2). For...both proteins.     Figure 2: Two primary antibodies with the same isotype can not be used for multiplexed... are the same isotype and therefore cannot be indirectly probed simultaneously on the sample. What to ...because the pair belong to the same isotype and will react with the same secondary. If you were to change...that group. If you use two antibodies that are the same isotype then the secondary antibody will bind to...

...detect your targeting vector or gene of interest. 2. Breed your parental mice to generate F1 Once you’...experimental and control mice will have been raised in the same environment and will harbor similar epigenetic modifications...multiple mutations is if the mutations are on the same chromosome. Homologous chromosomes recombine during...two loci, which would get both mutations onto the same chromosome. Until crossing over occurs between the...

...to (1) run a DNA gel to confirm fragment size and (2)  incubate with RNAse and Proteinase K to purify the...proteins of interest to any DNA they were bound to (2) fragmented the DNA (3) used antibodies to specifically...adjustments, to get everything just right!  Fig. 2: Goldilocks is actually celebrating a successful ChIP...you’re working with a tissue sample, you’ll need to homogenize your sample before moving on to the next...Homogenization for a tissue sample can also need significant optimization for the sample type and size.) It can...process is very similar for both, though tissue samples do require an extra homogenization step. For this...to focus on the ChIP process and not the upsteam sample collection process or the downstream analysis process...

...backbone alone; 2. Transformation containing ligation mixture with insert and backbone. Sample results indicative... used. Follow the manufacturer’s instructions.   2. Isolate Your Insert and Vector by Gel Purification...DNA concentration alone. One method is to conduct 2 ligations for each plasmid you are trying to create...colonies than the vector + insert plate (unsuccessful 2), or roughly equivalent numbers of colonies on each... or equivalent to backbone + insert, unsuccessful 2 and 3 above), you can try ligating the recipient plasmid...between samples. In addition to a DNA ladder standard, it is also a good idea to run an uncut sample of each...you to either stain your gel after you run your samples or add the stain as the gel is being made (post...

...Currently available from the Kim lab are 2 presynaptic and 2 postsynaptic targeting mGRASP plasmids. Additionally... cells were pulsed with 2µg/ml of Doxycycline for 2 hours, and imaged on the Keyence BZ-9000 fluorescence...work! Robinson et al., Dev Cell 2010 Feb 16;18(2):324-31. Cheeseman et al., J Cell Sci 2013 May 1...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...

...two genes over time, at a range of distances from 2-75 Mbp. Labeling multiple, intrachromosomal loci Building...successfully resolved loci with physical map distances of 2 and 75 Mbp, with the calculated fluorescent distances...established physical map. In comparing pairs of targets ~2 Mbp apart, they noticed that they could evaluate the...dCas9 supplied by a separate vector. Figure 2: CRISPRainbow generates 6 unique colors and a white... Find plasmids from this publication at Addgene. 2. Ma, Hanhui, et al. “Multicolor CRISPR labeling of...plasmids from this publication at Addgene. 4. Kiani, Samira, et al. “Cas9 gRNA engineering for genome editing...

...possible when each of the XFP genes is introduced at ~2 copies per cell per color, following a Poisson distribution...(prepared with Addgene #104110 - #104112) (Figure 2). We’ve also created constructs with containing chemical...AAV titer - The XFP genes have to be introduced at ~2 copies per cell per color to achieve the highest color...not powerful enough for large brain samples. For large brain samples, we recommend pretreatment with ScaleCUBIC1...time points, sacrifice the animals and fix the samples. When you use AAVs, it typically takes a few weeks...excessive amount of XFPs expressed. Then, clear the samples and visualize. For more details on this procedure..."Optical clearing and index matching of tissue samples for High-resolution fluorescence imaging using ...

...Request review: 1–2 days Prep and QC completed: 6–8 weeks Average shipping time: 1–2 days (USA); 3–4 days... our production process. This process will take 1–2 days, and we anticipate approving the vast majority...produce your Packaged on Request prep and run the same quality control assays we run with all of our in-stock... be 1x1013 and titers for AAV2 to be 7x1012, the same titers found in our in-stock preps. If a request...

..., AAV2/8 has a type 2 ITR and a type 8 serotype. Almost all vectors contain type 2 ITRs, which comes from...differentiated, non-dividing cells.   Figure 2: DNA rescue and transduction of a conventional single-stranded...quantitative PCR-based method for the titration of AAV-2 vector stocks."Molecular Therapy 6.2 (2002): 272. ...episomal and does not divide and replicate in the same way the host DNA does. Because of this, fast dividing...McCarty, D. M., Paul E. Monahan, and Richard J. Samulski. "Self-complementary recombinant adeno-associated...

...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):...cellular level in the mouse. 2003 PubMed PMID: 12665802. 2. Branda, CS and Dymecki, SM. Talking about a Revolution... SM. 2008. A FLEX switch targets Channelrhodopsin-2 to multiple cell types for imaging and long-range ...between the sites. If a DNA sequence is floxed in the same orientation, the SSR will excise the DNA fragment...reporter with one loxP site and one lox511 site in the same orientation, and placing the additional loxP and...

...digital PCR. Human Gene Therapy Methods, 25(2), Article 2. https://doi.org/10.1089/hgtb.2013.131 Wang,...(see maps section for images).     Figure 2: AAV Pro cells were transduced with either pAAV-Ef1a-mCherry-IRES-Cre...Digital PCR Method. Human Gene Therapy Methods, 29(2), 96–103. https://doi.org/10.1089/hgtb.2017.198   ...of the viral capsid proteins VP1, VP2, and VP3. Samples of viral preparations are subjected to polyacrylamide...proteins as a fraction of total protein present in the sample is also determined and used to determine purity...shown relative to the total protein present in the sample.   Sterility Viral vector preparations are ...rigorous barcode matching system that follows the sample from the DNA tube all the way to the cleared viral...

...those in Figures 2 and 3 don’t show these regions, even if they are present in the sample. Other techniques...Cas9 from Streptococcus pyogenes.   Figure 2:  Crystal structure of S. pyogenes Cas9 in the apo...NUC and REC lobes laying open like a book (Figure 2). The portion of the CTD that recognizes the PAM is...it doesn’t appear in the crystal structure (Figure 2) at all. When this crucial recognition region is disordered...itself at the bottom of the image. Stem loops 1 and 2 extend horizontally across both lobes. The guide RNA...is required for Cas9 nuclease function. Stem loops 2 and 3 are not strictly necessary for in vitro cleavage...open conformation, but highly flexible, as in Figure 2. The guide RNA is bound primarily by the REC lobe,...