...functions in mammalian expression systems.   Christopher Newgard’s Lab has fused LSD1 to the smaller nuclease-dead...specified loci. Charles Gersbach’s lab has deposited mammalian expression constructs including pcDNA-dCas9-p300...Tet1-CD for targeted cytosine demethylation in mammalian cells. This plasmid is used with pcDNA3.1-MS2-...DNMT3A-PuroR for targeted cytosine methylation in mammalian cells. Co-expression markers EGFP and PuroR enable...pcDNA3.1-dCas9-MQ1(Q147L)-EGFP, a fusion of dCas9 to a small DNA methyltransferase from the prokaryote Mollicutes...Jaenisch R (2016) Editing DNA Methylation in the Mammalian Genome. Cell 167:233–247.e17 . https://doi.org...

...i-GONAD is performed directly on pregnant female mice using a small incision that exposes the ovaries and ...into blastocysts to be implanted in pseudopregnant female mice. If you obtained the desired chimeric mice...template with 55-100 bp homology on each side. For optimal repair, place the loxP sites precisely at the cut...microinjection and i-GONAD knock-in efficiency using a small ssDNA, and they found comparable efficiencies (52...However, microinjection does require 2.5x more animals than i-GONAD and is a more technically difficult...for Cas9 Purification Find CRISPR Plasmids for Mammalian Expression ...

...calculated p values from smallest to largest and ranking them (1 being the smallest value). It calculates...test. Distribution - spread of data; typically, normal (bell curve) or skewed (see Fig. 2). Assumptions...   Parametric test assumptions: Distribution – Normal Variables – Independent Variance – Approximately... the assumptions for the tests described above (normal distribution, equal variance, and independent variables...histogram is sometimes sufficient to determine normality (or lack thereof). Alternatively, a Quantile vs...from a theoretical distribution. If your data is normally distributed, it will form (more or less) a straight...

...expression vectors are available at Addgene. A set of mammalian expression vectors containing various spike protein...nonstructural proteins 1 to 16, spike proteins, envelope small proteins, and membrane proteins. Although all viral...modification may not be easily expressed in non-mammalian cells. Yeast expression plasmids The yeast expression...cleavage, codon usage, and expression system. Mammalian expression plasmids Finally a set of wild type...transforms, innoculates, and performs DNA minipreps for small batches of samples. As soon as that process is complete...data! These same plasmids are then added into our normal full plasmid NGS queue in order to bring them inline...

.... To overcome this, most scientists use mutant animals that are genetically altered to overexpress endogenous...labeling (PL) uses a promiscuous labeling enzyme and a small-molecule substrate to map the spatiotemporal distribution...PL were APEX2 and BioID. APEX2 is fast, tagging proximal proteins within minutes, but requires H2O2, which...temperature range than BioID, which only functions optimally at 37℃. The increased efficiency of TurboID and...easily concentrated. Viral RNA is then amplified isothermally, and detected through a toehold switch, an RNA...the purified domains include protein stability, small molecule binding screens, peptide arrays, and phage...

...method, we recommend basing your selection on a small-scale head-to-head comparative study under conditions...calculated by multiplying EC by QY (EC*QY, sometimes normalized to the value of EGFP). The higher the number,...Determining practical brightness When a dish of mammalian cells is transiently transfected with a plasmid...in the fluorescence intensities of individual mammalian cells within the dish. This is caused by the stochastic...Another way to dodge cellular variation observed in mammalian cells is to tag an endogenous gene. By imaging...Mouridi et al (2017) and Heppert et al (2016). In mammalian cells this would be possible with CRISPR/Cas-based...

...This post, along with a future companion post on mammalian vectors, will catch you up on the core replication...cell structures similar to those of plants and animals. Unlike bacteria, yeast can post-translationally...CEN). These vectors replicate as though they are small independent chromosomes and are thus typically found... vectors are stable without integration. Yeast Episomal plasmids (YEp): These are most similar to bacterial... by the plasmid may be expressed at higher than normal physiological levels due to high copy numbers. ...and ability to be used in bacterial, yeast, and mammalian cells.   ...

...the full genome CRISPR screen approach is to use smaller libraries targeting subsets of genes. With such...where CRISPR modified cells are transferred into an animal and the behavior of the transferred cells is followed...of candidate genes from a full genome screen, a smaller, secondary, CRISPR screen could be used to validate...Addgene, and then use Green Listed to design a smaller validation screen using selected gRNAs extracted..., studying genes affecting cellular behavior in animal model systems. Use Green Listed to extract gRNAs...), Yusa (5), Wang/Lander/Sabatini (6), and Chari/Mali/Church (7). These reference libraries contain multiple...much, showing that we are far from consensus on optimal gRNA design. With Green Listed you can also easily...

...to the force to control protein expression in mammalian cells: easy to use biological tools are abundant...when knockouts aren’t viable). siRNA or RNAi are small, double stranded RNA molecules that degrade target...by placing tet response elements upstream of a minimal promoter. These systems come in two varieties: ...degradation. Since the tagged protein of interest is normally expressed, protein levels will quickly recover...the ligand is removed. Degron tag systems include small molecule assisted shut off (SMASh), auxin-inducible... and quickly degraded. However, if the relevant small molecule is introduced into the system, it will ...

... Their production requires animal sacrifice and because they are an animal derived product, there can ...limitation and allows for the flexible design and small scale production of antibodies. Problems with antibodies...Genetic Tagging - Since antibodies are produced in animals instead of via recombinant DNA, they can’t be genetically...and better penetration of the sample due to the smaller size of RANbodies compared to antibodies. Staining...Reporter-nanobody fusions (RANbodies) as versatile, small, sensitive immunohistochemical reagents. PNAS. PubMed...

...infected a non-small cell lung cancer cell line containing an activating mutation in epidermal growth factor...researchers to individually label millions of mammalian cells through lentiviral infection and to monitor... other replicate; prior to treatment, there are small (previously undetectable) populations of cancer ...combination treatment with multiple drugs reveals even smaller preexisting populations with resistance to both...distinct evolutionary paths to become resistant to epidermal growth factor receptor inhibition." Nature medicine...

...create a double-strand break works well because very small deletions and insertions in the DNA sequence can...nucleotides in length and lack an open reading frame, so small deletions or insertions may not result in a functional...degrade targeted lncRNAs. The Cas14 variant is a smaller and more versatile option. It can be used to target...specificity, particularly in applications requiring smaller delivery systems (Zibitt et al. 2021). Even though... Cas13 Targeting RNA with Cas13a (C2c2) Cas13d: Small RNA-targeting CRISPR enzymes for transcriptome engineering...10.1038/nature20149 Jiang Q, Meng X, Meng L, et al. Small indels induced by CRISPR/Cas9 in the 5’ region of...

...last updated Jul 12, 2021. What started out as a small collection of plasmids for coronavirus research,...They developed a method that uses loop-mediated isothermal amplification after reverse transcription (RT-LAMP... reaction to detect SARS-CoV-2 nucleic acids. Mammalian targets During the 2003 SARS outbreak, Hyeryun...the virus interacts with human cells. Addgene’s mammalian target plasmids express these and other targets...many expression systems: bacterial, yeast, and mammalian. These plasmids contain both untagged and tagged...2021.04.22.440848 Crawford KHD, Eguia R, Dingens AS, Loes AN, Malone KD, Wolf CR, Chu HY, Tortorici MA, Veesler D, Murphy...

...interaction between the fluorophores and serves as a small-scale ruler to measure distances that are too miniscule...orientation of the donor and acceptor proteins is not optimal, FRET may not occur or be detected even if the ...experimental setup as a potential source of error in suboptimal or undetectable FRET, well-characterized FRET...need. FRET biosensors designed to measure specific small biomolecules or gene activity are often intramolecular...interactions, measure the concentration or activity of small molecules, detect cellular processes and signaling... lead to monitoring of these processes in live animals (Hirata and Kiyokawa 2016), and molecular tension...

...immunization has its drawbacks: it requires a camelid animal facility which can be expensive to gain access ...advantages of synthetic nanobodies Nanobodies are small (12-15 kDa) single-domain proteins that can bind...because it contains two large heavy chains and two small light chains that stick out to form the “arms” holding...a heavy chain antibody (HCab). Because of their small size, nanobodies can be synthetically expressed ...without the need to find a llama farm! To find an optimally binding sybody, you want to start out with a large...protocol goes through 3 major steps to select for optimal sybody candidates. Figure 2: Outline of the...

...integrates into the genome and is propagated during normal DNA replication and cell division. Mobile-CRISPRi...right Tn7 end sequences. Donor bacteria with a chromosomal copy of the RP4 transfer machinery mobilize these...and recipient bacteria that lack an integrated chromosomal copy of Mobile-CRISPRi. For firmicute gram positive...tri-parental mating. Donor bacteria contain a chromosomal copy of the RP4 transfer machinery, which mobilizes...Enterobacter cloacae, a bacterium that’s part of normal human gut flora. Guides targeted essential and ...guides per gene. The library was grown in glucose minimal media with 100-fold excess of wild-type bacteria...

... easier for a smaller, recombined plasmid to overtake full-size plasmids (or for smaller plasmids to overtake...diversity in the library is maintained. However, for smaller libraries, chemical transformations can also be...using, as well as other factors, such as your lab’s normal transformation protocol, the size of the cuvettes...direct competition with other colonies containing smaller, recombined backbones that may replicate faster...on solid media. If your plasmid library is of a smaller size with a single size insert, liquid media can...

...plasmids used to inactivate specific genes in mammalian cells. Despite the popularity of CRISPR-based ...recombination occurs, the gene still functions normally because one of its exons has simply been replaced...been used to create many knockout cell lines and animal models, their efficiency is very low, ranging from...also offer hope for the creation of new knockout animal models in species that were previously difficult... rats. If you have tools for creating knockout animals or cell lines, please consider sharing them by ...Genes On and Off Resources on Addgene.org Find Mammalian Expression Vectors Find Cre-Lox Plasmids Learn...

...are read after each cycle. The raw data is then normalized to a reference sequence present in the same amount...housekeeping’ gene), and quantified relative to a 'normal' sample with known copy number set as the expression... all qPCR assays, samples, standard curves, and normal controls must be run in triplicate.  The nice thing...the amount of DNA in your sample relative to your normal control - you can set up your calculations so that...RQ values used to call copy numbers should be as small as reasonably possible and set using the variance...though it does not require a standard curve or a normal control, it can take a few hours longer than qPCR...

...such as malaria and dengue for many years. Since more than a thousand children died of malaria today, ... tropical mosquito vectors of diseases such as malaria and dengue are currently being performed in regions...because the drive must be much more efficient and minimally harmful in order to spread. Barrier confinement...should be used? There are ongoing efforts to develop formal guidelines to answer this question. Until then,... are those of the author and do not represent a formal stance taken by Addgene or its staff. Additional...

...genetic elements may differ (e.g., a set of various mammalian promoters), the same cloning procedure can be ...fluorescence imaging or flow cytometry applications in mammalian cells. The modules used in this method include...each containing a unique fluorescent protein with minimal spectral overlap between them (Table 1). In each...DNA sequences, no restriction enzymes needed Not optimal for joining sequences with a high degree of identity... applications in flow cytometry approaches in mammalian cell culture systems (their original goal). Their... for Imaging and Flow Cytometry Approaches in Mammalian Systems. PLoS One. 2015 Apr 24;10(4):e0124958....

... is even smaller than SaCas9 and is also compatible with AAV delivery. NmCas9, another small ortholog,...variants are capable of cleaving genomic DNA in mammalian cells and zebrafish embryos, and they can be used...Staphylococcus aureus (SaCas9) is about 1 kilobase smaller than SpCas9, so it can be packaged into adeno-associated...has been adapted for targeted RNA cleavage in mammalian cells. The REPAIR (RNA Editing for Programmable...JH, Kim J-S (2017) In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni...

...a no-fuss genetic knockout  Knock-out cells or animals are engineered by introducing Cas9-mediated frameshift...catalytic residue of a protein or knocking out a very small peptide. In these cases, you have little flexibility...(bases 4-8 of the protospacer). While this is a small window, this system can be used with a variety of... transcription by blocking its initiation. In mammalian cells, robust repression requires targeting dCas9...bacteria, yeast, plants, drosophila, worms and mammals (Browse plasmids by model organism, here).  More...delivery systems can be found here “CRISPR 101: Mammalian Expression Systems and Delivery Methods”. Validating...

...But CRISPR’s uses were still limited to small deletions and small insertions, leaving a several kilobase-sized...systems. Some of these Cas systems have enzymes small enough to fit into a dual AAV system, albeit with...similar to shRNA and Cas13 systems in a range of mammalian cell lines. However, in those same comparisons...came Prime editing — the ability to now make any small changes, including base edits, insertions, and deletions...mutations, all found in the same gene. If we only have small, directed editing abilities, any gene editing treatment...

...these small ligands. References 1. Ma, Hanhui, et al. “Multicolor CRISPR labeling of chromosomal loci...cleavage and an RNA aptamer that bound the compound malachite green (MG) to create a non-protein based visualization...Broccoli cassettes, thereby extending their use into mammalian cells. They’ve also dimerized Broccoli to further...that require recognition of ions or particularly small molecules that do not generate an immune response...imaging of RNA processing using broccoli reveals optimal aptamer expression strategies.” Chem Biol. 2015...

...expression in mammalian cells. This was the first codon optimized membrane protein expressed in mammalian cells...coli. Genes are also optimized for expression in mammalian cells. For example, Henry Lester’s lab optimized...Overall, codon optimization may not always be the optimal strategy for increasing protein production. If ...both determine and synthesize DNA sequences for optimal expression in your system of choice. ThermoFischers...Caenorhabditis elegans GluCl ion channel genes for mammalian cells dramatically improves expression levels....

...new genetically encoded calcium indicator using small monomeric NIR fluorescent proteins (miRFP670nano...calcium reporters iGECI and NIR-GECO2G, and its small size (~1500 bp) makes iGECInano suitable for AAV...et al. Design and Initial Characterization of a Small Near-Infrared Fluorescent Calcium Indicator. Front...two bright fluorescent proteins with different thermal quenching rates (mNeonGreen and tdTomato), they...variety of imaging conditions (155 μs and <0.1 °C in optimal conditions). While B-gTEMP itself is a significant...  Lu K, et al. Intracellular Heat Transfer and Thermal Property Revealed by Kilohertz Temperature Imaging...

...adding Late Embryogenesis Abundant (LEA) proteins, small proteins that naturally adsorb to the AWI, can protect...RvLEAMshort here!    Abe, K. M., & Lim, C. J. (2024). Small LEA proteins as an effective air-water interface...editing to rescue a genetic disease phenotype in an animal model. Find PE-eVLP plasmids here! The lab’s other...Efficient site-specific integration of large genes in mammalian cells via continuously evolved recombinases and...variants, including a “miniCRISPR” version, are much smaller proteins than SpyCas9, allowing them to be packaged...types in the nervous system By Ashley Waldron The mammalian nervous system is composed of myriad cell types...

...AAVs) make fantastic gene delivery vehicles for episomal gene expression and are particularly useful for...have been using AAV vectors for gene editing in mammalian cells since 1998. In that year, Russell and Hirata... titre, and are therefore able to infect only a small subset of the cells used in a given experiment. ...genome editing is hampered by the fact that even small Cas9 homologs like SaCas9 and their requisite gRNAs...engineering using adeno-associated virus (AAV)." Chromosomal Mutagenesis. Humana Press, New York, NY, 2015... AAV: A Versatile Tool for Gene Expression in Mammals AAV Titers: Where do they come from and what do...

...to be incorporated into the edited genome. In mammalian cells such as stem cells, HDR is relatively rare...that for generating a homozygous HDR event with optimal efficiency a guide RNA targeting a cut <10 bp from... other on each target site.   Figure 3: Optimal cut site to mutation distances for heterozygous...introduction of a desired sequence change by HDR, the optimal guide RNAs should not only have sufficient on-target...off-target activity, but should also mediate a DSB at optimal distance from the intended mutation site, depending...: 23287718. PubMed Central PMCID: PMC3795411. 2. Mali, Prashant, et al. "RNA-guided human genome engineering...