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...the usual transfection drawbacks: transient expression and needing to work with a transfectable cell line...Cassettes transfection gRNAs expressed from a polycistronic transcript  Requires co-transfection of Cas9...Cassettes transfection gRNAs expressed from a polycistronic transcript Requires co-transfection of Cas9  ...expression platforms: transfection, lentiviral transduction, and AAV transduction.  In most of Zetsche ... rely on transfection of more than one vector to express the gRNAs and Cas9. Co-transfections can lead... GFP tag were transfected on a single plasmid. This is versus 2.4% of cells transfected with a pool of...Lentiviruses carry a (+) strand RNA copy of the DNA sequence, which is a suitable substrate for Cpf1. This means...

...Reverse Transcriptase and Integrase components. Transfer cPPT Central polypurine tract; recognition...γ-Retroviral transfer plasmid encoding a transgene, sgRNA, or shRNA of interest: The transgene sequence is...packaging and transduction are not encoded by the transfer plasmid, but instead are provided in trans by other...integrated into the host genome upon viral transduction. Most transfer plasmids available from Addgene were ...packaging, and transfer) are supplied by three types of plasmids, which are cotransfected into the 293T...cell line. γ-Retroviruses can transduce target cells, but the transduced target cell does not produce ...proviral DNA synthesis. Increases transduction efficiency and transgene expression. Psi (Ψ) RNA target...

...considered. Q2: Can I use lentiviral transfer vectors for transient transfections? A2: Technically, yes. Will ...produced by co-transfecting three essential ingredients: the lentiviral packaging vector, a transfer vector including...specifically for transient expression (find some useful backbones here) or from viral transduction. While not...3rd-generation transfer vectors apart? A3: Addgene defines 2nd- or 3rd-generation transfer vectors based... Are transfer plasmids replication competent? What is SIN? A6: Most (if not all) Addgene transfer plasmids...enhancers and the transgene; such interactions can alter expression of not only the transgene but also that...which is rarely more than 30% of the transfer plasmid. Many transfer vectors are SIN. Packaging plasmid...

...., 2017), axonal transport  (Murlidharan et al., 2014), and postsynaptic transmission (Oh et al., 2020... S. (2013). Analysis of Transduction Efficiency, Tropism and Axonal Transport of AAV Serotypes 1, 2, 5... an Immune Response against the Tetracycline-Dependent Transactivator Correlates with Long-Term Doxycycline-Regulated...Doxycycline-Regulated Transgene Expression in Nonhuman Primates after Intramuscular Injection of Recombinant...adeno-associated viral vector-mediated transduction of the corticospinal tract: Comparison of four promoters. ...Specificity of gfaABC1D-Driven Transgene Expression in AAV-Mediated Gene Transfer. Molecular Therapy - Nucleic...great in a GABA neuron, for instance, may not transduce the adjacent norepinephrine neuron at all. There...

...their straightforward husbandry and ability to produce abundant, externally fertilized, transparent progeny...per individual to maintain. Also in contrast to mice, the transparent zebrafish embryo develops outside ...mRNA inhibit its translation either by blocking initiation or splicing. (RNAi, in contrast, engages existing...et al., 2019). Generating zebrafish transgenics Creating transgenic organisms by introducing foreign DNA...different experimental conditions. Strategies for generating transgenic zebrafish are well established (...Mosimann, 2016). One common and robust strategy uses a transposon called Tol2 to randomly integrate foreign...lineage tracing Over a period of 24 hours, the externally fertilized zebrafish zygote transforms into an...

...effects of the transfection itself (i.e., the vector used in transfection, transfection reagent, or transfection...experiments employ transfection, negative, positive, and replicate controls.  Transfection controls: empty...vector and internal control A transfection control measures transfection efficiency and enables observation... allows you to examine if the transfection reagents or the transfection process itself has any cytotoxic...Another type of transfection control is an internal control vector, which measures transfection efficiency....luciferase) that is either co-transfected with the test plasmid or transfected into a separate well of your...GFP-expressing Plasmid B, could demonstrate whether the cells were transfected successfully and expressing...

...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...

...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...