...RNA polymerase III transcript, the sgRNAs were encoded on the lentiviral minus strand in the 3’ LTR. These...HeLa cells transduced with SARS-CoV-2 nucleocapsid protein (Plasmid #141391-LV [+N]) or membrane protein ...high microglia transduction efficiency for the indicated ssAAV5s. Figure adapted from Serrano et al. 2023... with tRNAs, which are cleaved out of the RNA transcript by endogenous processing enzymes (Figure 1). ...including modifications to the Cas9 or reverse transcriptase enzymes, inhibition of host cell proteins, and...microglia are notoriously resistant to viral transduction. Through in vivo screening of human promoters...scAAV vectors have limited size compared to single-strand AAV (ssAAV). To maintain the packaging capacity...

...changing its double-strand cut to a single-stranded nick. In order to produce a double stranded break, two sgRNAs...place on the free strand of DNA and converts a C to U without producing a double strand break.   Prime... target DNA. The reverse transcriptase polymerizes DNA onto the target strand based on the sequence of...targeting dCas9 to transcriptional start sites was sufficient to repress transcription by blocking its initiation...approach is that two sgRNAs targeting opposite strands must be available in moderate proximity to each...desired edit adjoined to the sgRNA, a reverse transcriptase, and a nCas9. While this system has been shown...been exploited to localize factors such as transcriptional activators and repressors to target genes. ...

...small, double stranded RNA molecules that degrade target mRNA transcripts. When transiently introduced, ...fused to a transcriptional repressor to your gene of interest. It will inhibit transcription instead of...of introducing a DNA double strand break. Transient introduction of CRISPRi or inducible expression (more...LoxP vector.   Turn it off for a little while Transient suppression of expression is faster and easier...expression. Addgene has a dCas9 KRAB plasmid for transient knockouts!  Turn it on Turn it on forever Permanently...for integration at a specific genomic site, or randomly integrated (once or many times) into the genome...that, too! CRISPR activators (CRISPRa) are transcriptional activators fused to a dead Cas9. The system...

...and GFP reporters, as well as multiple transient and transgenic systems, Sahoo et al. showed that gene...commonly used 35S promoter. Transgenes are also efficiently inherited in both transgenic tobacco and Arabidopsis...tumor-inducing” or Ti plasmid, which allows it to transfer genetic material into the host plant genome. Scientists...Scientists have worked to optimize this system for gene transfer, studying the stability of modified Ti plasmids...a new and versatile binary Ti vector for both transient and stable gene expression applications in plants...borders specify the region of DNA that will be transferred to the plant, similar to viral vectors’ terminal...binary vectors are quite large (>10 kb.) Since transformation efficiency is inversely proportional to size...

...non-exclusive gene silencing pathways: transcriptional and post-transcriptional gene silencing (PTGS). In either...silence the target gene(s) either transcriptionally or post-transcriptionally. In plants, different dicer-like...where approximately 20-30 nucleotides of double-stranded RNA (dsRNA) results in downregulation, or “silencing...either pathway, short double-stranded RNA fragments (siRNAs) are produced from long dsRNA via an RNase III... through targeted mRNA degradation, blocking translation, or chromatin modifications of complementary ... Figure 1: Schematic of an RNAi-inducing transgene. Typically, the desired target of RNAi is expressed...alternative start codons, and/or the phenomenon of transcriptional adaptation (Smits et al., 2019; Sztal and Stainier...

...signal downstream of the transcription start site will terminate transcription of most of the ncRNA (Figure... coming, right? Non-coding RNAs (ncRNAs) are transcribed but do not encode proteins. They play important...Using a single guide RNA (sgRNA) to create a double-strand break works well because very small deletions and...sequence can completely disrupt expression of the transcribed ncRNA (Jiang et al. 2014).  Simple knock-out ...lncRNA regulatory elements that are crucial for transcription and downstream function. Rather than attempting...the lncRNA locus, scientists can mutate the transcription start site, splice sites, exons, and/or promoters...the removal of exons. Disrupting promoter or transcription start site function would also help to reveal...

...the zeptomolar range while One-step-SHERLOCK has a sensitivity in the femto-aptomolar range. For more details...are designed, each binding to one strand of the reverse-transcribed target sequence that are present close...reverse transcription. This is then amplified by PCR using Taq Polymerase and then transcribed to provide...LED and an orange filter to detect fluorescence upon cleavage. Highlights: Reverse-transcribed RNA samples... Suresh from Iowa State University. Since its appearance, SARS-CoV-2 has spread to almost every part of... milliliter can be detected – in the zeptomolar range Time: ~1 hour Equipment required: 37°C water bath...amplified. RPA reactions also contain reverse transcriptase to produce cDNA. Isothermal amplification is...

...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...XmaI restriction sites). Another way is to transform AAV transfer plasmids into bacterial strains, like NEB...Overview: The first step in packaging AAV is transfecting HEK293 cells with AAV packaging plasmids. The... into the virus. Two to five days after transfection, the AAV-containing cells and media are harvested...more details. Pro tips I’m not going to lie, AAV transfer plasmids are kind of a pain to work with. These...structures that are prone to deletion from the transfer plasmid. The good news is there’s a few simple...

...containing transcription factor binding sites can increase transgene expression and transduction efficiency...as the gene that they package. In a transfer plasmid, the transgene is flanked by two ITRs, and the ITRs...modifications can drastically affect gene transfer efficiency and transgene expression (Zhou et al. 2017). For...these mutations can affect transduction efficiency and subsequent transgene expression. Replacing the ...nucleotide sequences that flank the ends of the single stranded DNA genome of AAV in the native virus. In an AAV... the feature its characteristic T-shape. The arrangement of the B-B’ and C-C’ palindromic sequences determines...efficiency (Shitik et al. 2023). Transgene expression can be altered by using ITR sequences from different AAV...

...Transcription factors (TFs) act as the gate keepers for gene expression, turning transcription on and... regulatory network. The transcription atlas and library The transcription factor library The human...lentiviral particles and used to transduce human embryonic stem cells. Transduced stem cells were differentiated...approximately 1,800 TFs, with >3,500 unique isoforms transcribed from these sites. The Zhang lab generated a comprehensive... contribute to differentiation, the Zhang lab transduced human embryonic stem cells with the viral TF ...differentiation outcomes as well as global transcriptional changes. Using this road map, and what was ... uncharacterized TFs is a useful finding. Transcriptional program changes from this screen successfully...

...neurons! Neurons are really cool because they can transmit information, for example, from one of the 180 ...associated virus (AAV) shows relatively low retrograde transport efficiency in most serotypes. Some viruses (e....e.g., rabies) naturally exhibit retrograde transport and can therefore perform retrograde tracing, but...efficiently infects neurons, directs high-level transgene expression, and has retrograde functionality to...genetically controlled function. By using AAVrg in Cre transgenic mouse lines, you can access and manipulate an...from the hippocampus to Broca’s area would be transduced (Fig 3B). These specific neurons could then be...requires high titers for efficient retrograde transport. For recombinase-dependent vectors, this can cause...

...through transformation, so the cloned vector must be ligated. But in LIC, longer single-stranded overhangs...T4 polymerase (left branch); by annealing two separate PCR products (middle branch); or by incomplete ...step to retain products with single-stranded overhangs (right branch). The mixed PCR and incomplete PCR...overlapping regions of single-stranded DNA. But as long as the double-stranded region is long enough to hold...nicks easily repairable by the bacteria after transformation. LIC is a reliable cloning method, but it is... sequences (20–60 bp) on either end and single-stranded 5′ overhangs… but it doesn’t really matter if ...recombination intermediate to bacteria, so it can be transformed as-is into cells. The cells then get to work ...

...the transient cotransfection of the mARG plamsids into HEK293T cells using a chemical transfection reagent... These plasmids can be used directly in transient transfection in rtTA+ cell lines (such as HEK293-TETOn...into the genome by cotransfecting them with a plasmid expressing PiggyBac transposase. In rtTA-cell lines...engineering a cell line, cotransfect the mARG cassettes 2:1 (A:N-V) with PiggyBac transposase and outgrow the ... of replication (p15A) worked best under a wide range of inducer concentrations (0.1-1% L-arabinose) without...proteins. Excess gvpA plasmid added into the transfection mixture in HEK293T cells gave drastic improvement...improvement in expression. Stabilizing the gvpA transcript with WPRE reduced the number of copies of gvpA ...

...is codon usage important? Translation and protein synthesis relies on transfer RNAs (tRNAs). tRNAs bind...rate of translation and thus protein expression. In fact studies have shown that translational efficiency...on an organisms codon usage bias to increase translational efficiency and thus protein expression without...mammalian cells. This was the first codon optimized membrane protein expressed in mammalian cells. In this ...conformation, folding and stability, change post-translational modification sites and even affect protein function...Mauro and Chappell, 2014). Different rates of translation by different tRNAs, including those that exhibit...actually be critical for determining the rate of translation. The ribosome may slow and pause during elongation...

...concentration of FLEx vectors guarantees that most cells are transfected with all labeling plasmids and...PMCID: PMC2593125. 2. Buchholz, F, P O Angrand, and A. Francis Stewart. 1998. “Improved Properties of ... sites can lead to excision, inversion, and translocation of DNA in a similar fashion as between loxP ... has a temperature optimum of 37 °C (Buchholz, Angrand, and Stewart 1998). FLPe’s performance was further...in FLPO, which shows similar efficiency to Cre (Kranz et al. 2010). These differences in recombinase efficiency...-LDB domains of the fusions allowing Cre/FLP translocate to the nucleus where they exert their recombinase...brain? Cells of a specific brain area can be transfected by in utero electroporation. To achieve sparse...

...therapies and in vivo laboratory experiments ranging from transient gene expression in a particular cell type... Figure 2: DNA rescue and transduction of a conventional single-stranded AAV (ssAAV) and a self-complementary...genome conversion in the transduced cell nucleus, the single-to-double stranded conversion of the DNA goes...structure can readily fold into transcriptionally active double-stranded form through intra-molecular annealing...Adeno-associated virus (AAV) is a single stranded, Parvoviridae DNA virus, packaged in a non-enveloped...hijack host cell machinery, and synthesize a second strand of DNA before expression of their cargo can begin...integrate into the host genome and therefore the transduced gene is episomal and does not divide and replicate...

...for broad or narrow range of microbial species. These also contain the RP4 transfer origin as well as a..., the ability to detect mRNA transcripts of toxins and other transcripts of interest is a benefit of this...the organisms present while sequencing of all transcripts gives clues into what the microbes are doing....followed by a reporter gene. The hairpin blocks translation by sequestering the RBS, but binding of the “...microbial community) reveals the RBS and allows translation of the reporter gene resulting in GFP signal....lacks genetic tools is the bee microbiome. Nancy Moran’s and Jeffrey Barrick’s labs at The University of... RSF1010 origin, an IncQ origin with broad host-range (Meyer, 2009). The vector retains the oriT for delivery...

...generally are not transmitted to adjacent neurons via synapses (i.e., transmitted transsynaptically). This is ...retrograde transmission (Etessami et al., 2000, Wickersham et al., 2007). Thus, RABV transsynaptic spread ...replicate. Modified Rabies Virus: Converting a transsynaptic neuronal tracer into a monosynaptic (first order...subsequent cycles of viral replication and retrograde transmission occur, spreading the virus through higher and...higher order neural connections. While this transsynaptic spread is useful for neuronal tracing, it has...-dependent TVA to Cre-expressing neurons in a transgenic mouse line. By doing this, RABV expressing the...necessarily express EnvA unless it is supplied in trans. Monosynaptic RABV spread can be achieved by modifying...

...inducible gene expression. Transgenic and knockin mice As the name suggests, transgenic and knockin mice contain...their genome. While transgenic mice have the new gene construct incorporated randomly into the genome, knockin...Conversely, outbred strains were originally derived from random crosses between mice of uncharacterized backgrounds...several types of genetically engineered mice: transgenic mice, knockout mice, and mice with conditional...construct inserted at a specific site. To generate a transgenic mouse, microinjection of DNA or infection with...Non-homologous recombination incorporates this DNA randomly into the mouse genome. In contrast, knockin strains...animal for gestation. Nucleases that induce double-stranded DNA breaks, such as ZFN and TALEN, can used to...

.... They can be targeted to the plasma membrane to measure membrane potential and can also be directed to... the membrane), the voltage sensing component (i.e., rhodopsin naturally resides in the membrane), or ... of voltage gated ion channels all depend on transmitting and sensing electrical potential. Thankfully... want to monitor the voltage change across the membrane of cardiac cells. Similarly, neuronal signaling...propagated through multiple cells to do things like translate perceived signals to physical actions. Understanding...rhodopsin proteins were initially evolved for ion transport, not fluorescence, they are not nearly as bright...sensors contained a FP that localized to the plasma membrane due to the addition of several targeting domains...