...using CRISPR for generating mouse models Although CRISPR is incredibly useful for generating mutations by ...that efficient, often takes more than a year to generate a mouse model, and its success is not always guaranteed...mouse genome engineering From the very start, generating mouse models using CRISPR is easier than more...example, we often screen only 15-25 mice when generating knockout models with CRISPR and find that many...knock in with a donor oligonucleotide, we aim to generate 50 to 100 mice and are usually successful in deriving... process is much quicker. It takes 3 months to generate founder mice using CRISPR, as compared with 8 ...less. With CRISPR, it may cost about $5,000 to generate founder mice, while with conventional gene targeting...

...representative images, measuring the main outcomes in several different ways, and other questionable research... tested with a new experiment; and hypothesis generation, in which data are investigated to look for unexpected...trends or differences between groups. Hypothesis generation is critical for advancement, but we are too often...using hypothesis testing statistical tools that generate a p-value.  As an example, if you hypothesize ...clear distinction between hypothesis testing and generation, can be sure that results will be reported regardless...

...within and outside your field, and any number of interactions with professionals from other backgrounds at...simple, but they could in fact be interpreted in several different ways depending on the team. When Tony... from completing 2-3 large projects a year, on average, to completing 8-10 projects per year. Happier ...resources on the Addgene blog Read about other transferable skills Find all of our science career related...

... in a nonacademic direction, there are many “transferable” skills you have developed in addition to learning...your favor? Look for opportunities to practice transferable skills in ways that will also enhance your science... concrete things you can do to develop those transferable skills while you are also learning to be an ... transferable skills...

... mouse model generation easi-er! Generating mice before CRISPR The process of generating knock-in mice...unsuccessful. CRISPR made the overall process of mouse model generation simpler, as described in an earlier...Gurumurthy and Ohtsuka groups have obtained an average knock-in efficiency of 30-60%. This is much more...earlier post on generating CRISPR mouse models. For simple knockout alleles or point mutations, the 8-...conventional gene targeting (A), you must first generate your desired mutation in mouse ES cells, select...template. Figure 2: Overview of ivTRT to generate an ssDNA repair template. Although effective... most projects requiring only 40-60 zygotes to generate a correctly edited founder. Figure 6: Diagram...

...translating gene therapies from conceptual design to clinical trials involve identifying a therapeutic gene, finding...effective. Challenges for AAV gene therapy Although AAV-based gene therapies are promising, scientists are ...can interfere with virus-cell interactions and seriously hinder gene therapy outcomes. Anti-AAV antibodies...robust therapeutic outcomes. In contrast to other gene delivery tools that are under consideration, recent...Howe, and A. J. Thrasher. "Gene therapy progress and prospects: gene therapy for severe combined immunodeficiency...guest blogger Harshana S De Silva Feelixge. Gene therapy technologies hold great promise for improving or...thought to be incurable. Rapid advances in next generation sequencing technologies have allowed scientists...

... the important interactions. It’s also important to determine if the human interactors we found in our... that can be targeted (hopefully by existing therapeutics that are safe, effective, and already FDA-approved...describing a comprehensive SARS-CoV-2 virus-human interaction network in which we expressed 26 of the 29 SARS-CoV...proteins to fish for human “prey” proteins that interact with the virus. As a result, we identified 332...332 human proteins that the virus interacts with in cells. Because viruses rely on the help of human proteins...druggable) is a key step in our strategy to throw a therapeutic monkeywrench into the virus’ hijacking of human...the beginning of understanding how SARS-CoV-2 interacts with human proteins. For example, we used HEK293T...

...proteins’ interaction brings the pieces of the FP back together (Figure 1). This method is generally known...Split-TurboID for protein interactions (Alice Ting Lab 2020) Split-horseradish peroxidase (Alice Ting Lab...techniques are available to assess protein-protein interactions. One popular approach is to fuse a protein of...a qualitative or quantitative readout of the interaction in vivo or in vitro, and can be used for measuring...begin? Have no fear, Addgene is here to help!  General Design Figure 1: Two proteins are fused ...) and FP(1-10) fragments. When Protein A and B interact, the FP fragments can assemble the full structure...yield large (1-10) and small (11) fragments. In several cases an FP(1-10) fragment may be compatible with...

...next-door microbes which grow optimally in moderate temperatures (usually defined between a fairly nippy ... take bacteria out of their optimally suited temperature ranges and make them grow better at suboptimal...bitter cold.  Since our objective is to induce tolerance towards cold, we decided to focus on a special...cold-loving) bacteria. These bacteria inhabit cold temperature locales and psychrophilic enzymes help them survive... pH ranges, crushing pressures, near-melting temperatures, tons of radiation, or freezing cold conditions...materials degrade at a much slower rate under cold temperatures, and frigid conditions decrease the potential...minute quantity can significantly reduce the operational time for a given process. These are extremely...

...intense, but it is a general good practice to keep up with the scientific literature a little bit at a time.... Searching for literature My go-to search engines for finding scientific literature are Google Scholar...the literature until you have all of the information you need. Reading primary research literature The...with the literature There are a few ways to make sure you are keeping up with the literature in your field...you daily updates with relevant literature. Organizing the literature As a scientist, you will begin to...require numerous other references to scientific literature to substantiate the facts upon which it builds...means you have to become familiar with a body of literature related to the topic. Finding reliable references...

...binary protein interactions. However, FRET generally only tells you if two proteins interact and cannot be...purpose is by its protein-protein interactions (PPIs). These interactions are often modeled as binary relationships...additional interacting factors. Problems with Existing Methods for Studying Protein-Protein Interactions Difficult...proteins interact with the bait at a particular point in time, i.e. when the bait is interacting with a ...two bait proteins that are known to interact.  When this interaction occurs, BirA* activity is restored...proteins Y and Z, that are known to interact. When  proteins Y and Z interact, the two BirA* fragments complement...bait proteins interact, but labeling ceases when these two bait proteins stop interacting. This feature...

... protein interactions Learn about other types of fusion proteins Study chromatin interactions with CAPTURE...and makes it easier for you to map protein-DNA interaction with limited biological materials. Problems ...fixation step. This serves to stabilize DNA-protein interactions for subsequent sheering of DNA by sonification...capture weaker or more transient DNA-protein interactions, the reliance on fixation does increase the ...fact they are just non-specific or indirectly interacting. The CUT&RUN method Enter CUT&RUN, an enzyme-based...controlling the amount of cutting and mitigating noise generated by off target digestion. After MNase digestion...locus Browse FRET plasmids for studying protein interactions  ...

...now, phage therapy is not routinely done in most parts of the world. In the US, phage therapy is used as...it comes to administering phage therapy. The laws behind phage therapy, acceptable endotoxin levels, etc..., 2017 Facilitating the search of phages for therapy Phage Directory started out as a directory of phage... only used phage in basic research but not therapeutically: not all labs in Phage Directory have the resources...information and potential for successful phage therapy, Sacher and Zheng also saw the need to archive ...

... connecting each genotype with vulnerabilities) to design a therapeutic strategy that works. But, those... plasmid toolkit, the Broad Institute Target Accelerator Plasmid Collection, that we hope researchers ...and cancer phenotypes. Broad Institute Target Accelerator Plasmid Collections To overcome this first challenge..., we’ve developed the Broad Institute Target Accelerator Plasmid Collections, now widely available to ...assays, we figured that they would need to be generalizable and capable of measuring a variety of molecular... and morphological profiling can be used as generalizable strategies to map the function of rare cancer...exciting plasmid collections (together with the data generated from these alleles in the above publications),...

... the target cell’s surface proteins and this interaction determines the specificity and efficiency of ...different approaches to open science, we aim to accelerate the rate by which sharing speeds science.   Check...

...Targeting in Retinal Gene Therapies 10:17-14:34 : Progress in Treating Macular Degeneration and Other Retinopathies...Retinopathies 14:39-25:59 : Strategies to Make Gene Therapies More General (more than correcting a defect in a single...for Delivering Multiple Genes to Cells for Gene Therapy 28:40-35:10 : GFP Activatable Tools Being Developed... gene therapy...

... eighth generation of adenine base editors, called ABE8 (Gaudelli et al., 2020). The generated ABE8s needed...important attribute for therapeutic application. ABE8s demonstrate an overall improved base editing capacity...primary human cells. Now, scientists at Beam Therapeutics have found a way to improve editing in primary...systems, ABE7.10 (and the starting point for a new generation of ABEs), consists of 3 components:  a deaminase... and Head of Gene Editing Technology at Beam Therapeutics, used a previously developed bacterial selection...showed comparable editing behavior to ABE8.x-d. Overall, ABE8s showed a median increase of 1.94x editing...research! Base editing in primary human cells  Cell therapy approaches can use base editors to correct mutations...

...conversion of RNA templates to DNA but suffer from several important shortcomings including low processivity...processivity (ie. the probability of the polymerase continuing to copy the template rather than dissociating)....improvements to existing technologies based on polymerase processivity. Please find the MarathonRT plasmid...

...models for developmental processes and promising therapeutic tools. The two unique characteristics that define...differentiate into specialized cell types [2]. We general speak of 4 levels of stem cell potency: Totipotency...They are isolated mostly from abnormal and/or supernumerary preimplantation embryos that are produced during... in many organs in the adult human body. These generate specialized cells for their location, mostly to... bone marrow only differentiate into cells of peripheral blood. Typically, there is a very small number...cells can be used for different research and therapeutic purposes. Both types have their advantages and...isolated from the human body Usage Universal. Can generate all adult cell types. Limited. Only specific cell...

...protein-protein interactions that uses split fluorescent proteins tethered to potential protein interaction partners...been generated for yeast (Kim et al., 2019) and fruit flies (Bischof et al., 2018), the generation of such... answer — i.e., do two proteins interact or not? — FRET is preferable when the temporal dynamics of PPIs...Ahmed AW, Spinelli L, Brun C, Basler K, Merabet S (2018) Generation of a versatile BiFC ORFeome library for...used to detect the inter- and intramolecular interactions that underlie protein function. However, FRET...quantities (and the correct stoichiometric ratios) to generate useable data. What’s more, measuring and quantifying...method exists for visualizing protein-protein interactions (PPIs): Bimolecular Fluorescence Complementation...