...experimental design considerations used in all behavioral studies. General considerations for behavioral ...behavioral testing As a general consideration for behavioral testing in a laboratory, a high level of experimental...It is a very old branch of science, dating back several hundred years, with historical ties to psychology...given some time to adapt to a testing apparatus. General stress levels of the test animal may also be reduced...handling for a few minutes by the experimenter several days preceding the experiment. Handling can however...mouse breeding colony) Environment Olfaction, temperature, humidity, noise level, circadian effects (inactive...inactive versus active phase) Experimenter Interaction should be minimal and strictly controlled; optional...

...candidate genes! Disease model generation CRISPR has made the generation of genome edits accessible, fast...select and generate physiologically relevant models. In complex diseases, it can aid in generation of multiple...respond to an existing therapy. In theory, CRISPR could even be used to generate patient mutations and ...cancer and identified several candidate inhibitors of gene X in a screen. You generate a gene X knockout ...design. Alternatively, a disease model can be generated, and drugs screened for a change in pathology....identification of potential targets by CRISPR can accelerate the drug pipeline. High-throughput CRISPR screens..., gene knock out, etc. – CRISPR can be used to generate cell line or animal models which have the precise...

...employees at Dimension Therapeutics, she helped build and grow the gene therapy division of the company...journal Molecular Therapy. Vandenberge is also the Grousbeck Family Chair in Gene Therapy, Director of the...Khandan Baradaran’s expertise in viral safety, gene therapy vector development, vaccine development, and assay...Addgene. She was Head of Quality at Dimension Therapeutics, which was acquired by Ultragenyx Pharmaceutical...Baradaran met Addgene Executive Director Joanne Kamens several years ago through the Healthcare Businesswomen’...Stegmeier is the Chief Scientific Officer at KSQ Therapeutics and is an Addgene depositor. KSQ uses a CRISPR-Cas9...the first lentiviral microRNA-based system for polymerase II-regulated RNA interference. In the first two...

...and anterograde transport suggest that there are several parallel mechanisms in place (for review see Maday...retrograde tracing, albumin protein labelled with horseradish peroxidase (HRP; Kristensson and Olsson, 1971... Virus (RABV) which moves retrogradely from a peripheral site of infection to the central nervous system...it can replicate and by doing so spread across several synaptic connections (Ugolini et al., 1989). When...Lo & Anderson, 2011). In the past two decades, several virus species have been modified and used as tools... the viral proteins VP1, VP2, and VP3. To date several hundred variants, or serotypes, have been found...et al., 2008; Watakabe et al., 2015). There are several reports of both retrograde and anterograde intracellular...

...Monoclonal antibody drugs are popular therapeutics for a plethora of disease conditions, from cancer ... they elicit an immune response from the body. Several factors contribute to the immunogenicity of a drug...response. T cell epitopes are found in either the therapeutic antibody itself or as impurities in the formulation... algorithm to evaluate the immunogenicity of therapeutics in silico. These algorithms are trained on vast...score (Mattei et.al., 2022).    Figure 1: Generation of immunogenicity scores by predictive algorithms...algorithms fail to capture. In vitro evaluation of ‌therapeutic immunogenicity is typically performed using traditional...traditional 2D assays with immune cell lines or peripheral blood mononuclear cells (PBMC) in 3D models of...

...Advances in CRISPR/Cas-based Gene Therapy in Human Genetic Diseases.” Theranostics. https://doi.org/10.7150/thno...possibility to treat human genetic diseases, and several treatments are currently being tested in clinical...discovery/development of genetic targets and RNA therapies. [6]. In 2014, we wrote a blog post on CRISPR’...researchers are now using it to investigate potential therapeutics for complex genetic diseases. So we’re revisiting... showed that CRISPR/Cas9 could be used to edit several autosomal-dominant mutations that have been connected...s team noted that CRISPR/Cas9 has been used to generate mutations in a wide range of organisms, but its...stem cells in organoid cultures are stable,” said Gerald Schwank, first author of the cystic fibrosis report...

...basic immunology, cancer-immune cell interactions, cancer immunotherapy, as well as new approaches to reprogramming...tumor-associated inflammation for therapeutic benefit ”as stated by the organizers. My overall impression from this... discussed the effects of starvation on cancer therapy. Findings in previous animal studies suggested ...immunosuppressive reaction in order to improve therapeutic outcomes.  If you are studying inflammation and...visualization techniques it is possible to follow the interactions between tumor and immune cells in real time.... UCSF showed some impressive videos of these interactions (Ref 3). You can find useful tips on in vivo...to help rid us of cancer in combination with chemotherapy. To dive deeper into this field read our recent...

...The key is T4 DNA polymerase, which has 3′→5′ exonuclease activity. T4’s polymerase activity can be thwarted...For one thing, those 5′ overhangs can be generated in several ways. One option, imprecise T4 digestion...the target vector. 5′ overhangs can be generated by T4 polymerase (left branch); by annealing two separate...work directly on PCR products, with 5′ overhangs generated either by annealing a mixture of products or from...concentrations of insert to succeed. 5′ overhangs are generated in the linearized vector by T4 digestion; then...adjacent fragment or vector. 5′ overhangs are generated on all the fragments and the vector, specifying... this limitation, one option is to perform a hierarchical assembly, assembling fragments in multiple steps...

..., such as chemotherapy treatment or ionizing radiation, can also result in dsRNA generation. While these... Here, we will cover how these structures are generated, why it’s important for cells to sense their formation...these viruses have one thing in common – they can generate dsRNA as part of their infection and/or replication...inherently double stranded, while ssRNA viruses can generate dsRNA intermediates as part of the viral replication...mRNA. These RNA tools have been used in clinical therapy to target pathogenic proteins, as well as in the...as long/short interspersed nuclear elements can generate dsRNA by bidirectional transcription or as inverted...is synthesized from a DNA template, inherently generating RNA:DNA intermediates in the process. These transcription-coupled...

...complementary site in the genome where it will proceed to generate a blunt DNA double strand break. Cas9 has been...Cas12’s primary difference from Cas9 is that it generates staggered breaks, also known as sticky ends. Cas12a...targeting experiments requiring single strand DNA generation, such as homology directed-repair. Cas12a is ...outlined below as well. Cas12a also has significant therapeutic promise due to the fact that it is smaller than...simplicity of Cas12a makes it easier to package for therapeutic delivery by AAV, which has strict capacity limitations...sequences of Cas12a to further optimize it for therapeutic and laboratory usage. Fast facts – PAM requirement...TTTN. Best for: homology-directed repair or therapeutic delivery.   Cas14 – Mini, but single strand ...

...such as p300 and CBP histone acetyltransferases or CARM1 methyltransferase. This attendant signaling feature...years evolved from chloramphenicol acetyltransferase to luciferase, the basic assay remains widely used...Both physiological processes are controlled by deliberate and compulsory actions instigated by the central...central nervous system signaling to peripheral effector organs, which then return information such as nutritional...communication of such signals between central and peripheral organs because of their established roles as ...available or being developed to treat a multitude of therapeutic indices, including hypertension, cancer, diabetes...action). Primary or core coregulators (those that interact directly with NRs) exist in steady-state complexes...

...challenge for CRISPR HIV-1 therapies is sequence specificity. Whereas antiviral therapies target HIV-1 at the...Today, HIV-1 can be controlled with antiviral therapies such that the virus is undetectable in the blood...demonstrated two potential strategies for CRISPR-HIV therapeutics - one using dCas9-SAM to activate HIV-1 transcription...usually seen in healthy individuals. Antiretroviral therapy (ART) can virtually eliminate plasma HIV-1, improving...binding sites increased activation of HIV promoter-luciferase constructs. In multiple HIV-1 latent T cell lines...the probability of off-target effects. In such a therapy, CRISPR/Cas9 would need to 1. excise the HIV-1 ...most HIV-1 patients. Obstacles for CRISPR HIV-1 therapy Both of these methods represent exciting advances...

...labs were also able to see movement and interaction between several different immune cells. These methods...to track cells from one organ to another. Thus several labs were able to decipher the journey of lymphocytes...low-energy photons (typically from the same laser) cooperate to cause a higher-energy electronic transition...molecule (see figure to the right). The excitation generated by these two-photons occurs only at a chosen focal...immune cells within organs and visualize how cells interact after a particular stimulus. Using an epifluorescence...quantitative measurements of cellular motility, interactions, and response dynamics. In the past 15 years...two-photon microscopy has uncovered host-pathogen interactions leading to the understanding of effector function...

...stearothermophilus grows at temperatures ranging from 30-74 °C, with an optimal growth temperature of 55 °C. For a...GeoCas9 is that it functions at temperatures much warmer than SpCas9 can tolerate. Harrington et al tested GeoCas9...at elevated temperatures. In both cases, GeoCas9 outperformed SpCas9 at high temperatures. See Figure ... These Cas9 proteins don’t work well at high temperatures, so to use them in thermophiles, bacteria must...CRAA (R = A or G) Spacer Length 20 nt 22 nt Temperature Range for Optimal Activity 35 - 45 °C 50 - 70...activity or longevity. GeoCas9 is active at high temperatures Figure 1: Thermostability of SpCas9 vs...GeoCas9 RNPs were incubated in reaction buffer at temperatures ranging from 25-80 °C. Activity is expressed...

...CRISPaint toolkit includes several donor plasmids for protein tagging, including luciferase (NanoLuc), fluorescent...lab developed a polymerase chain reaction (PCR)-based system that allows users to generate a plasmid containing...is expensive and time consuming to individually generate gRNAs and homology arm repair templates containing...scientists a convenient one tube assembly reaction to generate high-throughput targeting vectors. These vectors...includes a prebuilt donor plasmid library containing several types of fluorescent (EGFP, tagRFP, mCherry) and...donor plasmids from the library can be used to generate functionally unique repair templates. A final ... be easily modified to tag hundreds of genes, generating powerful genetic screening libraries. Additional...

...developed for each individual pooled library; generalized amplification protocols tend not to work as well... inserts of different sizes?), and the desired coverage for the library (How important is it that every...important factor to consider is your desired library coverage, since this will help decide what transformation...protocols aim for somewhere between 30x and 200x coverage, meaning that you need 30 to 200 times as many... your library. As an example, if you want 100x coverage of your library, and your library contains 5,000...thousands, if not millions, of plasmids, they are generally electroporated into electrocompetent cells to ...advantage is that chemical transformations are generally cheaper and easier. One library that Addgene distributes...

... mutations, the risk is high. You are generating a gene therapy product with CRISPR to (hopefully) be ...experiments where they can’t be tolerated. Below we will outline several options to quantify these events... or nothing to worry about all. Let’s consider several scenarios below for a CRISPR experiment which involves...-out edits in a gene(s) of interest and has an average off-target frequency of 5%. Your experiment involves...significant impact on your data. You are trying to generate a knockout cell line, with the goal of isolating... downstream experiments. You only successfully generate 1 null clone and proceed with it. In this case...event efficiencies. In addition to these tools generating a prediction for off-target levels, most of them...

...human gene therapy. Molecular Therapy: The Journal of the American Society of Gene Therapy, 14(3), 316...the viral particle sneaks its way into cells. Generally, AAV serotypes have a primary cellular receptor...strains of one species, so be sure to check the literature and validate your choice of AAV vector for your...AAV1 was the first viral vector approved for gene therapy (Issa et al., 2023). It may be a good choice for...central nervous system, including neurons, AAV5 is generally the best choice for targeting astroglia. It has...rAAV treatment, which can pose problems for gene therapy applications. Cross-reactivity of AAV serotypes...AAV7, and AAV8 in the Mouse and Rat. Human Gene Therapy, 19(12), 1359–1368. https://doi.org/10.1089/hum...

...including ours at Addgene, the overall purpose of a data pipeline is to perform several sequential computational...enabled us to rapidly iterate and optimize our pipeline. After reviewing and testing several platforms for engineering...parallels between Seqera’s mission and our own, as both address the need to accelerate science using different...sequencing at our facility using Illumina MiSeq Next Generation Sequencing (NGS) technology. As our repository...search for solutions led us to a partnership with Seqera Labs.              DNA sequencing drives our bioinformatics...sequences. More than half of these sequences were generated by Addgene as part of our QC process. Automation...other programs running on a computer, so it safely operates in isolation, and can be easily shared with others...

...survive high temperatures (up to 95 °C) and is active over a range of elevated temperatures (37–75 °C)....any fragments end-to-end without the exo/polymerase generation of annealed complementary regions. Why ...elevated temperatures, allowing further discrimination by incubating the ligation at a temperature near the...Reaction Temperature Calculator can be used to estimate an appropriate incubation temperature when using...restriction cloning), adaptor ligation for next generation sequencing (NGS) library construction, and circularization... does fidelity mean for a ligase? DNA ligases generally prefer fully Watson-Crick base-paired dsDNA substrates...as a correct base pair. DNA ligases have been generally found to have a higher discrimination at the upstream...