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...species. Existing protocols can transfer CRISPRi into a single bacterial strain, such as a B. subtilis, or ...bacterial Tn7 transposon system. This system has two parts: a plasmid expressing the Tn7 transposition genes ...chromosomal copy of the RP4 transfer machinery mobilize these plasmids for transfer to recipient cells during...: A Tn7 transposon containing the CRISPRi components and a plasmid with the Tn7 transposition genes are...are transferred to a recipient bacteria strain of interest by tri-parental mating. Donor bacteria contain...disease. However, the rate of transfer was variable. They measured transfer rates by comparing the number... P. mirabilis, had only ~1 transfer per 1,000,000 colonies. Higher transfer rates are better suited for...

...most cases, interested in transferring genes to mammalian cells (i.e. transferring DNA), yet retroviruses...still direct the translation of proteins because they integrate this reverse-transcribed DNA into the host...genome and then use cell machinery for transcription and translation. Here, we will discuss several aspects...worthwhile to generalize the idea of “transduction” to mean any transfer of material from the virus to the...a turn of phrase — “pseudotransduction” — to describe the direct protein transfer of RT, which would actually...After uncoating, reverse transcription follows the release of the two strands of viral RNA and of the ...structural proteins expressed from a reverse-transcribed piece of DNA called a “provirus.” This DNA is...

...engineered combinations of transcriptional activators to induce gene transcription at genomic sites targeted...p65-HSF1 transcriptional activator to each genome-bound dCas9 (see Figure 1a for the transgenic construct...different untranslated or coding regions. Typically, each gene isoform has a distinct transcriptional start...relatively straightforward to use CRISPRa to selectively target these different transcriptional start sites...distinct transcriptional start sites, all of which yield the same BDNF protein. To demonstrate the specificity...investigation. Co-transduction of primary rat neurons with dCas9-VPR and sgRNAs targeting either transcript variant...are often many more genetic strategies) and then consider whether traditional cDNA overexpression is suited...

...information from a region (anterograde tracer, Fig. 1B). Tracers are typically delivered to a particular... either trace downstream from the soma (neuron cell body) toward the axon (anterograde tracer) or upstream...making them useful tracers, but finding a virus that works well for retrograde tracing can be difficult....naturally exhibit retrograde transport and can therefore perform retrograde tracing, but often at the cost ...expression.     AAV retrograde Unlike traditional dye-based neuronal tracing reagents, AAV particles require...not be transduced at all by this serotype. Nichole Beebe in Brett Schofield’s lab demonstrated that pathways...neurons! Neurons are really cool because they can transmit information, for example, from one of the 180 ...

...Reverse transcription of the transgene occurs in the cytoplasm, then the resulting double-stranded DNA transgene... transgene is incorporated into a large complex of both viral and cellular proteins and trafficked to .... However, IDLV genomes are transcriptionally active, leading to transient expression of gene products...mutagenesis (Howe et al., 2008). 2. Transient expression of transgenes/shRNAs If you want your cells to ... for stable expression of the transgene. This makes IDLVs an attractive choice for certain types of clinical...quantitative PCR titration of vector DNA in samples harvested 24 hours post-transduction (Wanisch and Yáñez-Muñoz...They rely on a viral protein called reverse transcriptase to convert their RNA genomes into DNA to be ...

...modify a given gene. CRISPR-tranposons By combining CRISPR editing and transposons, the Sternberg and Zhang...developed CRISPR-transposons. The Sternberg lab's system called INTEGRATE (Insertion of Transposable Elements ... CAST (CRISPR-associated Transposase) both enable gRNA-directed transposition. INTEGRATE consists of four...multi-spacer CRISPR array, CRISPR-transposons can be multiplexed. Transcriptional repression (CRISPRi) in bacteria...s lab demonstrated that a minimal, regulatable CRISPR system effectively silenced transcription of one...repressed or activated transcription of select genes in E. coli. Furthermore, to demonstrate wider applicability...proteins involved in the activation of transcription including transcription factors, phage proteins, and RNAP...

...editing techniques. Inbred and outbred strains Mouse strains used in research can fall under one of two.... Common inbred strains include C57BL/6, Balb/c, and 129. Conversely, outbred strains were originally ...genetically diverse gene pool. Outbred strains are strategically bred to maintain maximum genetic heterozygosity...population. Outbred strains of mice are useful for comparing dominant and recessive traits and their impact...inducible gene expression. Transgenic and knockin mice As the name suggests, transgenic and knockin mice contain...DNA randomly into the mouse genome. In contrast, knockin strains are generated via homologous recombination...and provides a way to track successfully engineered cells. Similar to transgenic mice, knockout mice are...

...PA-FPs come in 2 flavours- monomeric and tetrameric. Tetrameric PA-FPS are better for whole cell imaging...By generating PA-FPs fused to intracellular proteins, scientists can track single protein localization,...localization, turnover, and trafficking, as well as organelle trafficking and dynamics (fission, fusion, etc). For...PubMed Central PMCID:PMC4007248. 11.Hess, Samuel T., Thanu PK Girirajan, and Michael D. Mason. "Ultra-high... proteins that display unique changes in their spectral properties upon exposure to a specific wavelength... wtGFP normally contains a mixed population of neutral (protonated) and anionic (deprotonated) forms of...generates a chromophore population that is more neutral, causing PA-GFP to display almost no fluorescence...

...structures in RNA transcripts Effects on transcription (remember, it’s not all about translation - e.g. codon...codons encode single amino acids. Evolutionary constraints have molded which codons are used preferentially...in any organism. So what are the evolutionary constraints that lead to these preferences and what can we...therefore may need fewer resources for the entire translation process. For example, during high growth rate... tRNAs. For example Tuller et al. found that translation efficiency is well correlated with codon bias...tRNAs, different regions of the protein may be translated at different rates. The ribosome will move quickly...the ribosome stalls, this may give the swiftly translated regions a chance to fold properly. For example...

...resonance energy transfer (BRET). The fluor then emits light at a tissue-penetrating wavelength above ...and imaged after i.p. or i.v. administration of the furimazine substrate for NanoLuc luciferase. Both ...luminescence. Intracellular Calcium Sensor Yang, J. et al. (7) wished to trigger intracellular Ca++ release...imidazopyrazinone substrate. ACS Chem Biol 7, 1848-57. PubMed PMID: 22894855. PubMed Central PMCID: PMC3501149...cloning with labs around the world and had specific strategies and tactics for getting the plasmids he wanted...group as well as evolution of the coelenterzine substrate to the more stable yet brighter furimazine by ...can generate autofluorescence and have limited penetration due to absorption by tissues. To avoid absorbance...

...sites). Another way is to transform AAV transfer plasmids into bacterial strains, like NEB Stable. NEB Stable...proteins to process a double-stranded intermediate into the single-stranded genome that's then packaged...viral replication; and 3) the transfer plasmid which encodes a transgene of interest that’s packaged into...particles for efficient gene transfer. I figure it’s better to have a bit extra AAV than to have to produce... iodixanol gradient ultrafugation Overview: Iodixanol gradient ultracentrifugation uses a gradient of ... share some AAV production, purification, and titration tips, while also summarizing the basic steps and...Overview: The first step in packaging AAV is transfecting HEK293 cells with AAV packaging plasmids. The...

...Figure 1f). Labeling strategy pros and cons All of these labeling strategies have trade-offs between the ...proteins. In-cell labeling strategies The majority of in-cell labeling strategies have two parts: (1) a genetically...(Figure 1c). Click chemistry (e.g., transcyclooctene–tetrazine)– Nonnatural amino acids can be incorporated...labeling strategies and improved fluorophores are making chemical dyes increasingly attractive and accessible...once exclusive to small-molecules: red-shifted spectra, ion sensitivity, photoactivation, etc. These impressive...before bleaching. The amount of information one can extract from a biological sample is wholly dependent on...requires more photons. For example, moving from transient overexpression of protein fusions to gene-edited...

...First Edition, trans. About CRISPR 101 en español January 2024 | First Edition, trans. About Una traducción...and management, resources for identifying your transferable skills, and tips on the job application process...

...General expression mRNA Tetracycline response element promoter Inducible with Tetracyline or its derivatives... low basal activity and several tetracycline operators. Transcription can be turned on or off depending...for in vitro transcription, the promoter drives either the sense OR antisense transcript depending on ...expression through varied inducer concentrations.  Sp6 in vitro transcription/ general expression Promoter...This element is responsible for initiating the transcription of your insert into RNA. In practice, the term...

...have to make a transgenic fly in order to study it. The process is fairly straightforward. Basically, you...you need to combine multiple transgenes in one fly, having the transgenes in different loci allows for...brief introduction as to how you can engineer new transgenic flies to study your favorite gene (YFG).  Sometimes...plasmids at Addgene Choosing a vector to generate a transgenic fly There are a few important points to consider...that you can control exactly in which locus your transgene ends up (1): Random insertion has the advantage...you can get varying expression levels of your transgene in different fly lines. Also, if there is a chance...for genetic recombination so that the transgenes can be inherited on the same chromosome. Targeted Insertion...

...Transcription, translation, and prone to degradation – those are the words that describe RNA! Double ...in our cells in the single strand form, but double stranded RNA and RNA:DNA hybrid molecules both exist...arise from a ssRNA engaging one strand of a dsDNA molecule; the 3-stranded structure produced is known as...intermediates in the process. These transcription-coupled R-loops are transient in nature and will dissociate...dissociate when gene transcription is complete. However, transcription of thousands of genes is constantly occurring...engages one strand of a dsDNA molecule, it does so at the expense of the unpaired DNA strand. A ssDNA molecule...Double stranded? Well, that’s just for DNA – right? RNA performs almost all of its biological functions in...

...sample of known concentration and measure a specific response as a function of the concentration. You then ...plot the concentration of the standards versus the response and use that information to extrapolate the concentration...spectrophotometer and concentration versus absorbance is plotted for the known concentrations (standard curve... a function of concentration on a scatter plot. For the BCA example, the concentration of each serial ...the trendline is calculated, you can extrapolate the concentration of the unknown sample based on its measured...calculated. The concentration of an unknown sample is determined by using the trendline to extrapolate its x value...standard curves with ELISAs to measure allergen concentrations in food; and your friend the grad student relies...

... could infect. Finally, the transfer plasmid encoded the desired transgene flanked by HIV-1 long terminal...integration. After co-transfection of these plasmids, 293T cells released transgene-containing lentiviral...protein), Pol (polymerase), Tat (viral transactivator for transcriptional activation from the 5’ LTR) and Rev...capacity of a lentiviral vector but for which transient transgene expression is sufficient. Lentiviral vector... PubMed Central PMCID: PMC110254. 3. In-vivo RGB marking and multicolour single-cell tracking in the adult...include a large genetic capacity and the ability to transduce both dividing and non-dividing cells. Lentiviral...integrate into the genome, permitting long-term transgene expression, but they could only infect actively...

... challenging to track intracellularly, but aptamers permit the study of intracellular RNA dynamics. Aptamers...Fluorescent RNA aptamers are used to track cellular processes by transforming cells with plasmids encoding aptamer-tagged...its ability to track RNA dynamics in a model of Fragile-X tremor/ataxia syndrome (Strack et al., 2013)....target cellular RNA to specific intracellular locations using Cas9 and track the localization of the complex...quantify RNA transcription by RNA Polymerase III. By expressing Corn under promoters for transcripts uniquely...PubMed Central PMCID: PMC5602116. Song, Wenjiao, et al. "Imaging RNA polymerase III transcription using...1187. PubMed PMID: 28945233. PubMed Central PMCID: PMC5679246. Strack, Rita L., Matthew D. Disney, and Samie...

...optimized your transduction conditions, you’re ready to transduce! Addgene has a transduction protocol you... the pooled library is transformed into E. coli for amplification. Transformed E. coli are allowed to ...functional titering and optimize transduction efficiency. Transduction efficiency can vary with titer, ...cells to transduce, you may want to consider spinfection. Spinfection is the process of transducing cells...cells at a concentrated seeding density while centrifuging. Spinfection transduction efficiency can vary ...reagent concentration.  Screening time Phew! You are now (finally!) ready to use your transduced cells ...this post, I’ll walk you through how to prep and transduce your pooled library.  Pooled libraries Scientists...