... PRKN mTurquoise2 CMV Parkinson's Pablo Rivera-Fuentes 111723 pBacMam2-DiEx-LIC-N-flag_huntingtin_full-length_Q23...

...attB sites, the phage integrates into the bacterial genome flanked by two new recombination sites (attL-left...reactions. Recombination of attP and attB sites creates attL and attR sites.   Gateway vectors contain modified...takes place between the attB sites flanking the insert and the attP sites of the donor vector. This reaction...enzyme mix and generates the entry clone containing the DNA of interest flanked by attL sites. As a byproduct...reactions. The BP reaction creates an attL-flanked entry clone. The LR reaction creates an expression clone ... place between the attL sites of the generated entry clone and the attR sites of the destination vector...attB, P, L and R sites that recombine very specifically and directionally: attB1 sites react only with ...

...and astrocytes Mice No (LY6C1)   Hanlon et al., 2019 AAV-BI-28 CNS: neurons, astrocytes, oligodendrocytes.... It can also effectively transduce astrocytes or oligodendrocytes when paired with appropriate cell type-specific...28 has been tested in C57BL/6J mice and effectively transduces NeuN+ neurons and astrocytes (Huang et al...et al., 2023). Among the capsids tested, AAV9-X1.1 showed the greatest fold change in RNA and DNA levels... others have done, you’ll need to experimentally test your chosen capsid before using it in an application...elements [promoters and enhancers, miRNA target sites, and recombination target sequences, denoted by ...cisterna magna or lumbar spine. Image and caption courtesy of Challis et al., 2022. Where do they come...

... Cre but lox511 sites can only recombine with other lox511 sites, not with loxP sites. Alternatively, ...at target sites to induce site specific recombination events: Cre recombinase binds loxP sites, while FLP... FLP binds FRT sites. When a DNA sequence is flanked by target sites (floxed) in opposing orientations..., you could use a pair of loxP sites and a pair of FRT sites and include both Cre and FLP recombinases...proceed either by first utilizing the loxP sites or the lox551 sites. Regardless, the first recombination step...fragment using either loxP or lox511 sites, leaving two identical sites on one end of the DNA fragment (Figure..., and that the orientation of the flanking loxP sites directs the Cre recombinase to invert, translocate...

...recombination one of lox2272 sites will now be sandwiched between 2 loxp sites and is excised by Cre (Figure...orientation and one of the unused (loxp) sites will be flanked by two lox sites now in the same orientation. Next...recombination sites, they hypothesized that spontaneous recombination between these sites may be the cause...leaky expression. To test their hypothesis, they randomly scrambled one pair of lox sites and either used ...injection site. Test the promoter It is also essential that the promoter be thoroughly tested in your cells...fibrillary acidic protein (GFAP) for astrocytes it is important to test it in the brain region of interest...transgene is floxed by two pairs of recombination sites and the open reading frame is inverted in relation...

...do some testing or ask experienced colleagues.   Figure 1: The laser pulse illuminates the cell... the next gates fully depend on your individual experiment. Practical example: lymphocytes in a mouse...first three gates out of the way, it’s time to move on to my experimentally-specific gates. Two-dimensional...free to use multiple individual gates. Note that in this case, square gates were used.  Congratulations...distinguish single cells from duplicates. Lastly, you learned which gates should always be included in ...samples, it is necessary to set up a sequence of gates to be able to select and precisely measure your ...in cell size, marker expression, and viability. Gates are the parameters the machine uses to differentiate...

...location and orientation of the loxP sites: Inversion: If the loxP sites are on the same DNA strand and are...region of DNA between the loxP sites is reversed.   Deletion: If the sites face in the same direction, ...If the sites are on separate DNA molecules, a translocation event is generated at the loxP sites. How can...limitation of using loxP sites is the inability to tightly control which loxP sites recombine if more than...GtATACAT). These variant lox sites undergo recombination with other sites of the same type, but are not...inter-molecular recombination at the loxP recognition sites. A key advantage of the system is that Cre acts ...experiments. LoxP (locus of X(cross)-over in P1) sites are 34-base-pair long recognition sequences consisting...

...between the sites (Fig. 1B below). Alternatively, if an attB-attP pair is anti-aligned (sites facing opposite... their recognition sites, or by excising one or multiple of their recognition sites from the register..... Figure 1D below demonstrates an example of a register that enters distinct states when treated with ...recognition sites, terminators, promoters, and genes) were built with flanking primer binding sites (designed...registers. The primer binding sites can also be used to design primers to read states by qPCR or Sanger sequencing...that recognizes its own attB and attP recognition sites on the register and catalyzes their recombination...state of the RSM. If an attB-attP pair is aligned (sites facing the same direction) on a register, then the...

... with three coordinates: chromosome, start, and stop. For instance (chr3, 1, 10) indicates a stretch of...notation works? The + operator denotes intersection and the – operator denotes set difference. That’s the ... critical NANOG binding sites: 1. Find Enhancers that have NANOG binding sites 2. Design a CRISPR screen...all of the DHS sites from 125 different human cell types from the ENCODE Project. DHS sites are the most...as a kind of “GPS for the genome” to compute coordinates for genetic elements like protein, RNA, and DNA...DNA (Table 1). As with GPS in the real world, coordinates alone aren’t very useful. We’ll need something...come in. They help us to transform raw genomic coordinates into meaningful experiments. Table 1: An emerging...

...recognizes FRT sites and initiates site directed gene recombination between those sites. FRT sites and loxP ... of target sites that differ in their sequence. Recombination between the two target sites with identical...and the interior target sites. In a second step, the two compatible target sites are cleaved out, leaving...loxP sites differ at the nucleotide level but share an overall structure of 13 base pair palindromic repeats...bp asymmetric core. Recombination between these sites can lead to excision, inversion, and translocation...translocation of DNA in a similar fashion as between loxP sites (For details see Cre-lox blog post). Optimizing ...systems in your experiments. Mutated FRT and loxP sites in Flex Vectors – inversion only once All recombination...

...considerations for behavioral testing As a general consideration for behavioral testing in a laboratory, a high... some time to adapt to a testing apparatus. General stress levels of the test animal may also be reduced...AAV5 targets both neurons and astrocytes, and AAV4 targets mostly astrocytes. Lentiviral vectors are pseudotyped...weeks before the behavior test. This time window between injection and testing will vary depending on whether...influence on the outcome and cause variability in the testing group. For example, if an experimenter picks up... will likely move around faster in the following test and skew results measuring locomotion. Mice may ...reduced by daily handling for a few minutes by the experimenter several days preceding the experiment. Handling...

...bits (1.375 bytes) of information. This leads to 2048 (211) possible combinations of states. Each of the...protein binding sites and 24 copies of the MS2 bacteriophage coat protein binding sites to tether many ...reporter proteins that can be used to test diverse secretory routes in various conditions. The plasmids ...enzymes, such that the ligation regenerates the original restriction sites for further iterative cloning...2xNLS) and c-terminal (Cas9(C)-2xNLS) fragments facilitates target DNA cleavage upon co-expression and spontaneous...which act irreversibly on their cognate attB/attP sites with no cross-talk. These pairs are closely related... in various combinations, while the cognate att sites are supplied as individual reporter constructs or...

...intronic regions and two repair templates in order to insert two loxP sites. Since two double-strand breaks...about 3 months. Image courtesy of Wenning Qin. Single-stranded DNA repair templates Short single-stranded...2 loxP sites to intronic regions 0.5 - 0.8 kb apart. In the example below, we’ll add loxP sites for conditional...disrupting splice donor/acceptor sites, Miura et al. (2018) recommend targeting sites at least 100 bp away from...place the loxP sites precisely at the cut site for each guide. This design also separates the gRNA target...single-stranded DNA templates have been shown to improve the efficiency of homology directed repair, with researchers...services or kits, including the IDT Megamer service tested by Quadros et al. When delivering the ssDNA template...

...cutting at mismatched guide sites, but not necessarily binding to these sites. Thus, these enzyme swaps ... ChIP-Seq, where only DNA sites bound by Cas are sequenced, has been tested with some success. The major... sequencing Sites with high sequence similarity to the gRNA can be sequenced. These sites can be selectively... this often means DNA cleavage at unanticipated sites. While off-target effects are nearly impossible ...or is engineered not to cleave DNA and instead activates/represses gene expression by various mechanisms...words, they can sometimes localize to unintended sites with sequence similarity to the on-target site. ...When a CRISPR-Cas system localizes to unintended sites and performs its programmed function there, this...

...behavioral tests.    Forced swim test The forced swim test has been used since the 1970s to test the efficacy...general considerations for performing behavioral tests, but now we’ll focus on how viral vectors have changed...locomotion, anxiety, and depression.  Locomotion tests To study and quantify locomotion, scientists monitor...Such a behavior can be monitored in an open field test (Alpar et al., 2018). Locomotion can also be used...therapeutic approach against drug addiction. Anxiety tests Rodents have a natural preference to stay in dark...neurons turns on overexpression of IL-1 beta. They tested the mice in a series of behavioral tasks. One of... of antidepressant treatment in rodents. In this test, an animal is placed into a tank of water with no...

...the effect of the independent variable you are testing. Importantly, they demonstrate the functionality...associated with the independent variable you are testing and not some other unintended factor. Control plasmids...luciferase) that is either co-transfected with the test plasmid or transfected into a separate well of your...Regardless, the amount of reporter protein activity correlates to both the amount of DNA transfected into the... Plasmid B (as internal control) This result indicates that the transfection was not successful due to... and from different preparations of the purified test plasmid expressing the independent variable. There...technical and biological. In general, technical replicates can be thought of as "plate controls". They are...

...generated by the nickases; D10A creates 5’ overhangs and H840A creates 3’ overhangs in a PAM-out design...problem. D10A nickase outperformed both WT Cas9 sites tested by a wide margin, exhibiting >20% repair efficiency...Optimize your spacing D10A: nick sites separated by 37-68 bp H840A: nick sites separated by 51-68 bp Use...CRISPR nickases improve homology directed repair rates, and share their design rules for your next CRISPR...as examples for this post. The D10A mutation inactivates the RuvC domain, so this nickase cleaves only...Conversely, the H840A mutation in the HNH domain creates a non-target strand-cleaving nickase. Instead of...Mollie Schubert and Shuqi Yan designed experiments to test nickase preferences using HEK293 cells. They started...

...widespread testing is crucial in controlling this pandemic. Currently, the CDC uses RT-qPCR tests to diagnose...100% negative prediction rate. It is also the fastest test that has been developed so far.  A similar method... Highly similar to the SHERLOCK diagnostic test, this test varies in the sequence of steps and the Cas...and to do so, the WHO recommends one strategy: testing, tracking, and social distancing. With countries...diagnose COVID-19 and some serological tests to determine past exposure. However, the limited availability...general schematic of CRISPR/Cas based COVID-19 testing methods.   Rapid detection of 2019 novel coronavirus...:10-100 copies of RNA per microliter Time: ~40 minutes Equipment required: 37°C heat block and 62°C heat...

...expression piggyBac Excisible Medium Medium Generates transgene-free and vector-free cells Genome integration...mutations Adenovirus Non-integrating Low Medium Generates transgene-free and vector-free cells No genomic... Sendai Virus Non-integrating Medium Medium Generates transgene-free and vector-free cells No genomic...Plasmid Non-integrating, usually Medium Medium Generates transgene-free and vector-free cells Limited genomic... EBNA1 episome Non-integrating Medium Medium Generates transgene-free and vector-free cells No genomic... Minicircle Non-integrating Medium Medium Generates transgene-free and vector-free cells No genomic..., criteria used, etc.) can effect reprogramming rates and such a number maybe misleading. Integrating ...

...Subscribe to the Addgene blog to get all the latest hot plasmid updates!     Tol2 Gateway-compatiable toolbox...vectors that have standard cloning sites or gateway compatible cloning sites for easy cloning of your genes...variations in gene expression from these polycistronic cassettes could lead to comparable experimental results....compared different variants of the polycistronic cassettes and discovered reprogramming discrepancies, which... Klf4 isoform (KLF4s or KLF4L). Polycistronic cassettes that contained KLF4s had overall diminished KLF4...global gene expression patterns. Polycistronic cassettes that contained the KLF4L isoform had more robust.... The polycistronic piggyBac transposon vectors tested in this work are available at Addgene. Kim, et...