...off-target pokes to nearby organs. Restraint: scruffing Location: peritoneal cavity Gauge: 25–27 Volume...suspension to create xenograft models. Restraint: back scruffing or mechanical restraint Location: back or hind...but has a pretty low learning curve! Restraint: scruffing Location: throat Gauge (bulb-tipped feeding tube...to intermediate (tail vein) Intraperitoneal Scruffing Peritoneal cavity 25–27 < 2–3 mL Intermediate ... < 0.02–0.05 mL Beginner Subcutaneous Back scruffing or mechanical restrainer Back or hind legs; between...Anesthesia Nose n/a < 0.05 mL Beginner Oral gavage Scruffing Throat 18–20 (bulb tipped) up to 10 mL/kg Intermediate...

...typically a Cas9 H840A nickase fused to a reverse transcriptase) together with a prime editing guide RNA (pegRNA...containing a primer binding site and a reverse transcription template (RTT). The design of the pegRNA has... we performed high-throughput ‘self-targeting’ screens with oligo libraries where pegRNAs are coupled ...cell contexts. Figure 1: High-throughput screening of pegRNA efficiency and building prediction tools...sequence-based prediction models from self-targeting screens such as PRIDICT/PRIDICT2.0 provide valuable insights...your target. Figure 3: High-throughput screening of the effect of chromatin on prime editing efficiency...

...their coat protein to screen for binding to the target protein. The protocol describes going through two ...protein (sybody), ribosome, and mRNA. During the screening process, displayed sybodies that bind to the biotinylated...enriched pool of sybody candidates are then reverse transcribed into cDNA fragments that are efficiently subcloned...The enriched sybody pools from the phage display screens are subcloned (using FX cloning) into the expression...interest. The Seeger lab used the ELISA technique described in their protocol because it is less susceptible...

...plasmid, how will it be sent to me? Description: This describes how the plasmid or other product (kit...useful to me? Plasmid purpose: This is a brief description of what the plasmid was designed and used for...vector’s plasmid number here. Gene/insert: This describes what has been inserted into the backbone, and ...they’ll end up on the N or C terminus of the transcribed and translated insert. If it is an empty vector... sequencing. Growth in Bacteria: This section describes how to grow the bacterial strain in which the ...

...Unlike promoters which will create unique mRNA transcripts for each gene that is expressed, multicistronic...resistance gene. These reporters allow you to easily screen or select for cells that are expressing your gene...interest at high levels. Unlike vectors that express screenable or selectable markers from a unique promoter,...your gene as they are both derived from the same transcript. Of course multicistronic vectors do not have...efficient translation of multiple genes from a single transcript. The two strategies most widely incorporated ...incorporated into plasmids for research purposes are described below. IRES Elements Translation in eukaryotes usually...

...for transcriptional activation from the 5’ LTR) and Rev (facilitates nuclear export of transcripts). A...Enable Genome-Scale, Knockout Screening Read about Genome-Wide Screening Using CRISPR/Cas9 Additional...both for targeted modification and genome-wide screening.   Lentiviral vectors in the clinic Since lentiviral... vectors and genome-wide libraries for CRISPR screening. Sanjana NE, Shalem O, Zhang F. Nat Methods. 2014...

...functions such as epigenetic regulation and transcription by using locus-specific chromatin immunoprecipitation...worked extensively with enChIP systems. Can you describe this technology and its advantages? Fujii: In ...biochemical analysis of genome functions such as transcription and epigenetic regulation. To elucidate molecular...enChIP system using CRISPR technology. Can you describe this technology and some of its potential applications... any other technologies that you would like to describe?  Fujii: I believe that "Biology is Method", i.e...Hodaka Fujii lab's Addgene page and click the "Subscribe to Alerts" link in the Addgene Alerts box. ...

...biochemical properties. In 1995, Roger Y. Tsien described an S65T point mutation that increased the fluorescence...backbones for constructing fluorescent fusions. Transcription Reporter: Placing GFP under the control of a...promoter in a given cell type. This type of transcription reporting was among the earliest uses of GFP... gene of interest, or expressed from the same transcript as the gene of interest, but after an internal... GFP are available. Others: We’ve really just scratched the surface of the potential applications for ...to identify particular cell populations in drug screens, to visualize micrometastases in nude mice in cancer...

...expression method enables us to quickly screen and investigate transcription factor candidates and the regulation...technique that uses a virus and the plant's post-transcriptional machinery to silence genes of interest (Unver...use of introns, which will be removed from the transcripts in the plant but not from Agrobacterium. Test...transient leaf transformations are a great help in screening candidate genes and speeding up research. Certainly...Budak. "Virus-induced gene silencing, a post transcriptional gene silencing method." International journal...

...experience and skills would help you do the job as described. Let’s imagine you’re applying to a job that ... your cover letter, you can expand on that by describing how you developed and used a ddPCR assay to answer...start by looking at the requirements in the job description. For each requirement, ask yourself if you have...exactly what you would want to talk about! You could describe your organizational system, your rationale for...company/organization]. I am a [adjective that describes you] scientist with over [number] years of experience...the university]” Include keywords from the job description to help the hiring manager quickly make the connection...

...create secondary structures in RNA transcripts Effects on transcription (remember, it’s not all about translation...locations or fail to make it through the entire transcript resulting in the production of nonfunctional ...every amino acid I’d like to encode,” but, as described above, not every region of a protein should necessarily...translation - e.g. codon choice may interrupt transcription factor binding sites) As you might imagine,...algorithms and DNA synthesis companies such as IDT and GenScript host online codon optimization tools. Keep in ...

...protease allows a transcription factor to enter the nucleus and modulate transcription of the mCitrine ...your studies.  Find the plasmids to set up your screening systems at Addgene! Schematic of the evolution...their BioRxiv post, the authors generated and described several improved genetically encoded fluorescent...Takayuki Iwaki, Kimiko Hara, and Kazuo Umemura have described a simple method which alleviates the inclusion...activating expression of TCF4 while monitoring transcript and protein levels. Plasmids for multiplexed ...

...antibiotic’, very often we immediately imagine a prescription for a bacterial infection. While this post will...that the term ‘antibiotic’ can also be used to describe anti-parasitic drugs and anti-fungal drugs, because...development of resistance include (Figure 1): Over-prescription of antibiotics (for example, giving antibiotics...not finishing the whole course of antibiotics prescribed to them This may seem counter-intuitive considering...The take-home? Always take your medication as prescribed. How do we know which bacteria are resistant ...infection is still effectively treated. We can prescribe antibiotics for the shortest time period possible...

...They rely on a viral protein called reverse transcriptase to convert their RNA genomes into DNA to be ... larger protein: PR (protease), RT (reverse transcriptase), and IN (integrase). These viral packaging ...the retroviral life cycle for a more detailed description). Since the integrated portion does not contain... cycle initially proceeds as usual. Reverse transcription of the transgene occurs in the cytoplasm, then... McCray, 2010). However, IDLV genomes are transcriptionally active, leading to transient expression of...Lentiviral vectors tend to integrate into transcriptionally active regions of the genome (Schröder et ...

...SSC-H, or FSC-W/SSC-W, respectively. H, A, and W describe the shape of a histogram, which graphically represents... of the cell’s illumination (see Figure 2). H describes the maximum signal strength, while W results from...photocurrent vs. time. The resulting histograms is described through maximum current (H), the time the cell...depends on cell type and laser voltage. The voltage describes an electric potential that can be applied to the...comparing two markers simultaneously, we commonly describe the resulting populations as double positive (...that there are three parameters — H, W, and A — describing the intensity and duration of the detected signal...

... rounds of screening. Created with Biorender.com.   Some phage display libraries screen antibody genes...expression systems. scFvs are also small enough to be screened for with in vitro display methods such as phage...and VH sequences. Phage display also allows for screening scFv generated from the B cell antibody genes ...the ways antibodies are. scFvs are even used to screen for new antibodies. To do this, antibody variable...variable domain sequences are expressed as scFvs and screened with in vitro assays to select for strong binders...

... are isolated from the resulting colonies, and screened for the desired modification. Finally, the positive...final circular plasmid product pictured here. B) Screening of recovered plasmids for the mutation of interest...mutation). Similar results can be obtained with PCR screening using the primers specified by the arrowheads ... vastly facilitates the subsequent process of screening for succesfully mutated clones. Forward and reverse...can also form colonies under these conditions. Screening If a restriction site was introduced (or ablated...ablated), bacterial colonies can be screened by identifying the presence (or absence) of that particular ...

... Table 1 gives a brief overview of published descriptions of commonly used or emerging capsids. Selected...2017). AAV-PHP.eB has frequently been used for screening candidate regulatory elements such as enhancers...type-specific promoters or Cre lines. Spatial transcriptomics suggests that AAV-PHP.eB, when paired with ...capsid of choice for future promoter/enhancer screening. However, AAV-PHP.AX packages viral genomes at...target gene expression (EGFP, in the depositing manuscript) across the brain. AAV-BI-28 has been tested ...when using a ubiquitous CAG promoter. Spatial transcriptomics shows that its tropism is relatively more biased... Gradinaru lab. It was selected from a library screened in marmosets. AAV.CAP-Mac effectively transduces...

...from the event can be found below along with a description of the program beneath it. You can jump to the...the different time stamps in the description to watch any sections you’re particularly interested in, ...abbreviated methods section found within a published manuscript. Luckily, many organizations have been working...JoVe, and Bio-protocol all work to provide more descriptive and interactive protocols that make it much easier...would get in-principle acceptance of the final manuscript regardless of whether the ultimate results were...Minisymposium on Reproducibility Booklet - Including descriptions of many organizations promoting reproducibility...

...identify possible gene function. In this post, I will describe a useful and cost-effective method that our lab...and probe with live cells The following is a description of a technique published by Simpson, Sacher, ...be cloned into E. coli and subsequent proteins screened for cell-binding abilities (6). This can be accomplished...long-term library storage. After overnight growth, scrape up, pool and extract plasmid DNA directly from ...construction for functional genomic and metagenomic screening." Applied and environmental microbiology 74.5 ...Protein-Protein Interactions Use CRISPR For Genome-Wide Screens Resources on Addgene.org Visit Our Microbiology...