...in a clinical setting. CRISPR is often used for animal germline modification, to repair or add in disease-causing...recessive disorder affecting approximately 1 in 5000 males. DMD is caused primarily by frameshift mutations... reading frame, leading to the production of a smaller, but at least partially functional protein. Individuals... of the dystrophin gene would take place, with minimal risk of off-target editing. In order for the therapy...improvement, with only 30% correction needed for normal function. Even low-frequency editing could make...inflammation and necrosis compared to unedited animals. In grip strength, specific force, and other muscle...require HDR-mediated precision editing tailored to smaller patient populations. To bring DMD CRISPR therapy...

...Temperature Range for Optimal Activity 35 - 45 °C 50 - 70 °C Genome Editing in Mammalian Cells? Yes Yes ...tool of choice for a number of model organisms: mammalian cells, yeast, drosophila, plants, worms, zebrafish...at temperatures ranging from 30-74 °C, with an optimal growth temperature of 55 °C. For a brief comparison... experimental results. GeoCas9 RNP editing in mammalian cells Besides being able to take some heat, GeoCas9...GeoCas9 can introduce genomic edits in mammalian cells. Harrington et al tested GeoCas9’s editing skills...without the need to grow the organisms at lower than optimal temperatures. GeoCas9’s thermostability could also...

...AMA   Guidelines for giving feedback Use formal and informal mechanisms for feedback (more on this below...her management style. Formalizing the feedback session Giving regular, informal feedback is a very effective...to an event as possible. If you wait too long, a small correction can become an unnecessarily big issue...scientists seem to find it easier to give feedback in formal sessions using a specific list of questions after... it helps to plan effective next steps. For a formalized feedback session, answer the questions below ...

...tag or a fusion protein. Mammalian vectors One of our most popular mammalian expression vectors, c-Flag...the pcDNA series. It has the CMV promoter for mammalian expression, a multiple cloning site to insert ...cassette for growth and cloning in bacteria and a mammalian selection cassette to screen for positive transfected...transiently transfected cells, you will not need a mammalian selection cassette (e.g. 1435 pSG5L Flag HA). .... You can also upgrade your basic mammalian expression system and go for a lentiviral or a retroviral ...to study gene function. From making transgenic animals, gene targeting and genome modifications to reporter...

...lanes or a small portion of the gel.  What a waste?  I started looking for smaller and smaller gel apparatus...electrocution.  These days you can buy small gel boxes, but none as small as the baby gel box and this one ... I was wasting buffer running my tiny gels in a normal gel box. It took a bit more ingenuity to solve ...this problem.  None of the commercial boxes were small enough to minimize the required area and buffer ...

...samples were harvested after the behavior, or the animals being monitored were physically hooked up to invasive...whistles. The fibers are made from a process called thermal drawing, essentially where blocks of polymers are... – including microscale light emitting diodes, thermal sensors, and microfluidic channels. These features...temperature, study electrophysiology, and deliver small molecules.   Fun fact: A 1 cm block of polymer ...channels and can also record tissue temperature proximal to the implanted fibers. As important as its capabilities...device has a rechargeable battery, is relatively small, and can be detached easily whenever necessary. ... induce stimulation in target tissues of a live animal. They started by delivering an AAV construct expressing...

...molecules have a specific wavelength that they maximally absorb. The spectrophotometer measures these absorbances...contaminants. Nucleic acids (DNA and RNA) absorb maximally at 260 nm. Proteins on the other hand absorb best...nm and organic compounds and chaotropic salts maximally absorb at 230 nm. The A260/A280 ratio is used ...quantifying DNA using gel electrophoresis. Except, it’s smaller. And automated. Unlike gel electrophoresis, you...and the sample is separated by electrophoresis. Smaller fragments migrate quicker than larger fragments...fluorescent dye that intercalates into the DNA such that smaller fragments are quantified first before larger fragments...electrophoresis which shows fluorescence units over time. Smaller fragments migrate more quickly than larger fragments...

...AAV) is a small, single-stranded DNA parvovirus. AAV can infect humans and some other mammals but is not... allows it to replicate episomally, essentially remaining an extrachromosomal part of the host genome ...gamma-retroviruses have been associated with cancer in mammals, which is thought to be due to their propensity...cool tool in the laboratory! It is ideal for whole animal administration, such as in mouse experiments. AAV...which is non-enveloped. It can infect humans, many mammals, and even the avian population. Some adenoviruses...

...highly specific in mammalian cells without significant off-target effects, yet small enough to be packaged...Ladder Engineered miniature CRISPR-Cas system for mammalian genome regulation and editing Anti-FLAG Frankenbodies...favorite protein! While the target only requires a small epitope tag (FLAG is eight amino acids, HA is nine...x Engineered miniature CRISPR-Cas system for mammalian genome regulation and editing by: Rachel Leeson... the size of either Cas9 or Cas12a. Despite its small size, CasMINI is comparable in efficacy to dCas12a...can be done via a nuclease-active CasMINI. This small yet powerful system opens up new possibilities in...

...into our cells of interest. Although they have a small packaging capacity, AAV vectors were suitable for...response. Any investigator who is using an in vivo animal model should consider the possible elicited immune...immune response from viral infection. Animal safety is a top priority in our lab so we opted for AAV. ...serotypes may perform better (3). Brain Region  Optimal Serotype Striatum AAV5>AAV9>AAV8>AAV1>AAV6>AAV2...outlined in the table below. Cell Type Cortex Optimal Serotype Oligodendrocytes AAV8 > AAV2 & AAV6 ...desirable, transport can be reduced by injecting a smaller dose of AAV and using a serotype with weak CNS ...scAAV) (7). As single stranded DNA viruses, AAVs normally require second strand synthesis before gene expression...

...neuroscientists in studying circuits in laboratory animals and human tissue specimens. These technologies ...

...gRNA was present or b. when a gRNA binding a non-mammalian sequence (GAL4) was supplied. In addition to ...distances from 2-75 Mbp. Labeling multiple, intrachromosomal loci Building on the work of Chen et al., ...turned their attention to mapping pairs of intrachromosomal loci. The technique successfully resolved ...the degree of chromatin compaction even for this small distance! To the authors’ knowledge, this work represents... represents the first mapping of intrachromosomal loci, a major benchmark in characterizing the 4D nucleome...performed sophisticated tracking of multiple chromosomal loci in live cells. However, they note that certain...Hanhui, et al. “Multicolor CRISPR labeling of chromosomal loci in human cells.” Proc. Natl. Acad. Sci. ...

...applications, smaller affinity reagents means higher resolution. Many of the below are also small enough to...enough that their bulk can be a barrier at times. Smaller, slimmer affinity reagents can have deeper penetration... a suitable antibody has been identified but a smaller or less immunogenic version is needed.  Single-...likelihood of aggregation (Bates, 2019). They are small enough to be produced in bacteria and are generally... nature. Single-domain antibodies are extremely small at 12-15 kD but can have weak signals due to their...proteins, or DARPins, are genetically engineered small affinity proteins, around 14-15 kDs in size. These...

...was contributed by Neil J. McKenna and Bert W. O’Malley. Survival of all organisms depends on efficient...information such as nutritional status. Within the animal kingdom, this information is relayed through factors... receptors across multiple different tissues in normal and disease states, their circadian regulation,...Signaling Atlas (NURSA) at www.nursa.org. Bert O’Malley, M.D., is Tom Thompson Professor and Chair of ...Science 294, 1866-1870. (PubMed) Lonard DM and O’Malley BW. (2007). Nuclear receptor coregulators: Judges...Molecular Cell 27, 691-700. (PubMed) McKenna, N. J.and O'Malley, B. W. (2002). Combinatorial control of gene expression...coregulators. Cell 108, 465-474. (PubMed) McKenna NJ and O'Malley BW (2005) An interactive course in nuclear receptor...

...Vector Mammalian Lentivirus pRDA_512 (RSV-driven; lentiGuide-based) Mammalian Lentivirus... over 512) Mammalian Lentivirus pRDA_789 (CMV-driven; CROPseq) Mammalian AAV pRDA_889...AAV2) Mammalian Transposon pRDA_757 (Piggybac; constitutive expression) Mammalian Plasmid...features of your vector, which are: Organism (Mammalian or drosophila) Delivery type (lentivirus, AAV...

...constructs available at Addgene includes bacterial and mammalian mCyRFP1 or CyRFP1 expression plasmids, FRET constructs...method to increase CRISPR editing efficiency in mammalian cells. Their marker-free coselection, analogous...mutations in ATP1A1 can render cells resistant to the small molecule ouabain - and editing at the ATP1A1 locus...create SpCas9 base editors with editing windows as small as 1-2 nucleotides! To reduce off-target effects... HF-Cas9 (NGG)  Very low off-target activity; mammalian expression vector Rees H, et al. Nature Communications...looking for the production of ECT-DNA (extra-chromosomal telomeric DNA), which accumulates when T-loops...inhibit Spy Cas9 function in both bacteria and mammalian cells. These CRISPR inhibitors may provide a layer...

...your plasmid yield is sub-optimal? If you are consistently getting sub-optimal plasmid yields from your...Different plasmids and strains will vary in their optimal growth conditions. For many high copy plasmids,...therefore be cultured in media containing half the normally recommended antibiotic concentration. Add supplements... or more to achieve maximum plasmid yield. The optimal growth time should be determined for each plasmid...culture is getting the right amount of oxygen for optimal growth, as insufficient gas exchange will prevent...cultures in flasks may be shaken around 220 RPM, but smaller cultures, especially those in deep-well plates,...

... fluorescent proteins, chosen for minimal spectral overlap, minimal protein aggregation, and high photostabilty...Chemical approaches may require expensive peptides or small molecules and exceed a physiologically relevant ...Classic genetic engineering methods enabling chromosomal integration of sequences in bacteria are time-consuming... by otherwise pharmacologically inert drug-like small molecules, allowing labs to precisely and non-invasively...catalytically inactive Cas9 DNA-binding domain in mammalian cells. (Guilinger et al., Nat Biotechnol. 2014... different TALE scaffolds are provided, as the optimal scaffold can depend on the length of the spacer... in the mouse brain. Kim et al is utilizing a mammalian GRASP (GFP reconstitution across synaptic partners...

...them advantageous for small capacity vectors like AAV. In addition to the mammalian option described below... many tools to help you multiplex - we’ll use mammalian plasmids to introduce you to some of your potential...cleaved and released. These constructs tend to be smaller than constructs with multiple promoter-gRNA cassettes...available from the Yang lab for use in plants. The mammalian multiplex systems use the Csy4 RNA nuclease from...efficiency for point mutations and 66% efficiency for small insertions. Multiplexing of two targets simultaneously...Danilo, et al. “In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system.” ...modular vector system for rapid combinatorial mammalian genetics.” Journal of Clinical Investigation 125...

...cost-effective and faster-growing than mammalian cells, but mammalian cells can perform human-like post-translational... Y, Zhan C, Malm M, Field R, Turner R, Sellick C, Varley P, Rockberg J, Chotteau V. Small-scale bioreactor...can take place in bacterial, yeast, plant, or mammalian cells. Each of these systems has their advantages...bonds (Frenzel et. al., 2013). If you’re using a mammalian production system, cell lines typically used include... your plasmid(s), and then wait 7-14 days (in mammalian cells) to collect the cell culture supernatant...

...Yeast-based protein evolution Fluorescent tools for mammalian cells Nanobody purification CRISPRi and CRISPRa...affinity tags from purified recombinant proteins at small and large scale.  This new technique involves selection...tools for labeling structures and compartments in mammalian cells By Angela Abitua Genetically encoded fluorescent...from the Lck gene. For marking microtubules of mammalian cells, the author found that too high expression...label entire microtubules. Find these fluorescent mammalian cell structure markers plasmids at Addgene! Chertkova...antibodies can’t be overlooked. Nanobodies are smaller in size, more durable, and usually much easier ...the authors created the pMAK backbone by modifying pMAL-T-Avi-His/BirA, a vector commonly used for in vivo...

...let's review how E. coli normally replicate their chromosomal DNA. Chromosomal replication in bacteria ...synchronized with the replication of the host cell's chromosomal DNA.  Plasmids are said to be under stringent...replication fork. Termination sequences present in the chromosomal DNA ensure that the helicases are released from... replication.   Figure 2: Prokaryotic chromosomal replication. Adapted from BioRender template....regulatory mechanisms allow E. coli to control chromosomal replication and balance population growth against...

...affects primarily girls. After a period of seemingly normal development through ages 6 to 18 months, individuals...gene activation, and more. Loss of MECP2 in adult animal models results in rapid symptom onset, suggesting...influence Rett Syndrome. Zoghbi’s lab found that a small DNA binding motif in MECP2 interacts with the minor...disease that has shown reversibility of symptoms in animal models, Rett syndrome is a strong translational...delivery of a functional MECP2 gene in preclinical animal studies with positive results (Sinnett et al., ...programmable RNA editing can repair a gene in a living animal across multiple cell types using a mouse model ...repositories including patient-derived iPSCs, MECP2 animal models, tissue banks, Rett databases, and other...

... Learn All about Mammalian Vectors Read up on CRISPR Delivery Systems for Mammalian Cells Learn about...retracted due to cell line contamination: Epithelial Mesenchymal Transition Is Required for Acquisition of Anoikis...nitrogen store to prevent loss from equipment malfunctions. One Master vial can then be thawed and used...independent quality control for a range of testing, from animal doping to forensics, DNA sequencing to drug testing...PubMed PMID: 19722756. Additional Resources ATCC Animal Cell Culture Guide Thermofisher Guidelines for ... Resources on Addgene.org Find Plasmids for Mammalian Cells ...

...electrocompetent cells is minimal (picogram range). It can be extracted from small bacterial cultures (i.... approach, allowing scientists to clone from a smaller amount of DNA (ng range). To allow efficient and... Figure 3: Need to introduce plasmids into mammalian cells? Use transfection grade DNA. Transfection...the process of inserting an external plasmid in mammalian cells and it will likely require micrograms of...plasmid DNA, so that you can decide what will be the optimal type of plasmid preparation in terms of quantity...

...of interest is relatively small, adding GFP may alter diffusion rates. Smaller genetically encoded probes...cofactor binding proteins as described above. The smallest tags are peptides that are engineered to bind ... For a review see Lotze et al. (2016). To date, small probes that are entirely genetically encoded do ...in the medium or flavin containing proteins in mammalian cells. Generally, autofluorescence is less of .... "A genetically encoded fluorescent probe in mammalian cells." Journal of the American Chemical Society...bioluminescence imaging of deep tissue in freely moving animals." Science 359.6378 (2018): 935-93. PubMed PMID:...

...concentrations of template (e.g. 0.1-1.0 ng/μl). Smaller plasmids (~3 kb) are generally more efficiently... the unmodified plasmid will be split into two smaller bands (Figure B, digest A). In contrast, if your...digestion with the appropriate enzyme will merge two smaller bands visible in a digest of the unmodified plasmid...digest, which excises a short region (<400 bp) proximal to the target site, can identify these duplications... relative to the original template). Due to the small difference in size, the fragments should be separated...sequence), it may be easier to subsequently clone a smaller region (containing the specific modification) into...target site, you can simply amplify and mutate this smaller portion of the plasmid with a primer pair that ...

...’ll want to run gradients for optimal annealing temperature and optimal primer concentration; check for...parameters, some people find it can detect even smaller differences, and it can even be used to detect ...chemistries available and they can be the more optimal choices at times. For example, chemistries that... plate type or tubes, sealing method, and other small variations in consumables. Track and record these... passive reference dye, such as ROX, can help normalize for that. Passive reference dyes are inert fluorescent...machine surge errors and allow for well-to-well normalization of signal. They are often included in commercial... a protocol that automatically performs the normalization step.  Machines Most general-use protocols, ...

...to occur at a frequency of 1.2 per megabase in mammals), leading to DNA damage and developmental aberrations...indicating that the recombinases were inherited normally and thus nontoxic. In contrast, 0/79 offspring...two of these recombinases were also active in mammalian cells. In experiments, B3 and KD were shown to...site-specific recombinases for use in manipulating animal genomes. Nern A, Pfeiffer BD, Svoboda K, Rubin ... and DNA damage induced by Cre recombinase in mammalian cells. Loonstra A, Voojis M, Beverloo HB, Al Allak.../s004270100167. PubMed. Cryptic loxP sites in mammalian genomes: genome-wide distribution and relevance...

...targeted region, as shown below.  To identify optimal nickase designs, IDT scientists Mollie Schubert...consistently higher than that of H840A, especially with smaller nick distances. Using D10A, they subsequently showed...the PAM-out configuration, D10A tends to generate small deletions, while H840A is biased towards large insertions...and Yan next examined a situation thought to be suboptimal for HDR. At the AAVS1 locus, the two nearest ...13 nt away from the target site rather than the optimal <10 nt distance. Employing two gRNA sites that ...between nick sites Use 40 nt homology arms for small insertions or tags (ssODN; IDT Ultramer® Oligonucleotides...