We narrowed to 718 results for: des.1

...If a depositor provides a sequence for the plasmid, QC scientists will first align the de novo NGS result...experiments, to false conclusions.  Figure 1. Potential points where DNA errors can be introduced...are assembled into a circularized plasmid sequence de novo (without the use of a reference sequence) using...sequence, if one was provided. If no reference is given, de novo assembly in Geneious can still provide additional...plasmids in our collection. This standardization provides security for requestors, ensuring that the plasmid...types of data for each of their plasmids, from descriptions of key components of the plasmids to partial...believe may affect the function of the plasmid. This includes point mutations, frame shifts, missing elements...

...and Therapeutic Purposes. Annu Rev Virol. 2014 Nov;1(1):427-51. DOI: 10.1146/annurev-virology-031413-085355...personalized medicine. References and further reading 1. FDA approves gene therapy for blindness 2. Gene Therapy... Med Biol. 2014; 801: 687–693.  doi:  10.1007/978-1-4614-3209-8_86. PubMed PMID: 24664759. PubMed Central...whole field to a grinding stop and only now, 2 decades later, is it finally back in the limelight. On ...(such as viral vectors) without triggering a destructive inflammatory immune response. At the time, adenoviral...follow in the footsteps of Luxturna™. However, despite promising results in preclinical studies some potential...technology: engineering of new capsid variants, designing expression cassettes to regulate and optimize ...

... export license applications for VSV and SARS-COV-1 plasmids. Export control license process    An export...charged.  Timelines  We have offered VSV and SARS-COV-1 plasmids under this application process since May ...institution. Requesters will need to provide a brief description of the applicable research project when purchasing...

...NGS) to identify genes of interest. Figure 1: Overview of in vitro AAV CRISPR screens. Created ...certain experiments. AAV can be safely handled at BSL-1, while lentivirus requires a BSL-2 setup. This requires...CRISPR-induced DNA breaks. Nature Communications, 10(1). https://doi.org/10.1038/s41467-019-12449-2 Pupo,...diseases. Signal Transduction and Targeted Therapy, 9(1). https://doi.org/10.1038/s41392-024-01780-w Additional... This vastly increases the possibilities when designing screens. Each serotype has its own tropism, or...experiment. Additional considerations One of the downsides to using AAV is the limited packaging capacity...Adames, A. C., Saur, D., & Rad, R. (2022). Tutorial: design and execution of CRISPR in vivo screens. Nature...

... and materials required for each [1, 2]. This protocol [3] provides, in exquisite detail, the various ...follow him on Twitter @derekpsimonphd.    References 1. Gutierrez DV, et al. Optogenetic control of motor...manipulation of neural circuits. Nature protocols. 2012;7(1):12-23. doi: 10.1038/nprot.2011.413. PubMed PMID: ...freely moving mice. Neuroscience research. 2011;70(1):124-7. doi: 10.1016/j.neures.2011.01.007. PubMed ...

...unpublished, that allowed the visualization of specific de novo synthesized proteins in situ. I was privileged..., 2010) and allows the visualization of the whole de novo synthesized proteome. However, FUNCAT alone ... contact her at [email protected]. References 1. Dieterich, Daniela C., et al. "In situ visualization...radioactive materials is highly regulated, requires designated work areas and come with health risks. Importantly...synthesized endogenous proteins in situ. Now FUNCAT-PLA provides both quantitative and spatio-temporal information...

...Figure 1: Purify DNA using glass filters. Image: Kim and Morrison, 2009. This method provides DNA yield...a gravity-based column plasmid maxiprep kit takes 1-1.5 hours to complete, while this syringe-based method... solution. NaI has two purposes in this protocol: 1) it solubilizes both DNA and agarose, and 2) it helps...property and developed a protocol that uses Whatman No. 1 paper or even paper towels to purify DNA and RNA from...plasmid purification and DNA gel extraction method described in Kim and Morrison is nearly identical to that...

...identification of proteins associated with the IRF-1 locus. Figure 1: Schematic of enCHIP. Image from Fujita...the promoter region of the transcription factor IRF-1. Immunoprecipitation, using an antibody targeting ...References Campbell, Amy E., et al. "NuRD and CAF-1-mediated silencing of the D4Z4 array is modulated ...region In 2013, the Hodaka Fujii Laboratory first described a method to purify a specific genomic region using...plasmid. Experimental use Since the initial report describing CRISPR-mediated purification of a specific genomic...

...10.1038/s41551-024-01227-1. doi:  https://doi.org/10.1038/s41551-024-01227-1   CHARM: A compact epigenetic...spacer proteins ending with a GFP nanobody (Figure 1). This enrichment streamlines the purification and...highly-visible beads on the cryo-EM grid.     Figure 1: MagIC-cryo-EM for sample enrichment and structure...single-particle analysis of PRC2 with RvLEAMshort (1:6 molar ratio) with 10 minutes of glutaraldehyde crosslinking... A) Schematic of workflow. B) Detail of spacer peptides (3HB: 11-nm 3-helix bundle; SAH: 60-nm single ...cryo-EM samples from damage at the AWI. Plus, tardigrades!  Find RvLEAMshort here!    Abe, K. M., & Lim...

...2021).   Table 1: Properties of modern NIR FPs     NIR FP Ex, nm Em, nm ECa, M-1cm-1 QYb, %   Molecular...expression level. State-of-the-art FPs (listed in Table 1) were optimized for efficient binding of endogenous...previously reported widely used dimeric iRFPs (Table 1). In general, the monomeric state of a probe leads...iRFPs in cellular and molecular brightness (Table 1).  NIR reporters and biosensors NIR reporters can ...Biol 24, 758-766 e753 (2017). Baloban, M. et al. Designing brighter near-infrared fluorescent proteins: insights...

...lentiviral gRNA expression vector (pRDA_526) that (1) enables efficient knockout (KO) of genes in immune...statistical power of in vivo screens. Figure 1: Using FITS to perform in vivo CRISPR screens to assess...single gene KO and pooled screening approaches; (4) encodes a Thy1.1 selectable marker to facilitate the isolation...assess T cell responses and clonal dynamics in lymph nodes (LN) and tumor cells. Figure used with permission...also reveal novel pathways. The FITS deposit also includes pLX_305_Ovalbumin, a plasmid that enables introduction...

...the viral TF library, with each cell receiving only 1 TF expression construct. Cells were cultured following...states was generated (Joung et al., 2023). Fig. 1: Barcoded TF library was packaged in lentiviral particles...atlas of directed differentiation. Cell. 2023. 186 (1) 209-229. doi: 10.1016/j.cell.2022.11.026 Joung J....transcription factor library The human genome encodes approximately 1,800 TFs, with >3,500 unique isoforms... and screening mechanisms, although the unique barcodes for each TF help streamline identification. While...a disease in a model system. TF screens can be designed to identify custom TF programs to acutely initiate...

...reserve a small aliquot, split into two parts to (1) run a DNA gel to confirm fragment size and (2)  incubate...with a phenol-chloroform extraction.    Fig. 1: Schematic overview of ChIP. Image courtesy of Shengliu... let’s do a quick review. At this point you have (1) crosslinked your proteins of interest to any DNA ...immunoprecipitation (ChIP) is an extremely useful technique that provides insight into protein:DNA interactions. ChIP works...up the DNA, or enzymatic digestion. Sonication provides a truly random digestion, but is harsher and requires...both tissue or cell type, amount of protein, and desired fragment length for your output assay. A longer...

...dual-color control of neurons. Nature Communications, 12(1), 1–20. https://doi.org/10.1038/s41467-021-24759-5 Zhang...to consider when choosing one.     Figure 1: Example of an experimental optogenetic setup. A light... 21–51). Springer US. https://doi.org/10.1007/978-1-0716-0755-8_2 Lehtinen, K., Nokia, M. S., & Takala... 21–51). Springer US. https://doi.org/10.1007/978-1-0716-0755-8_2 Boyden, E. S., Zhang, F., Bamberg, E...In 2005, Boyden et al. described the first light-activated tool for controlling neuronal activity, channelrhodopsin... light in combination with retinal) variations designed to give researchers flexibility in using optogenetic...precise timing of the perturbation. Other tools are designed to be activated within a narrower band of wavelengths...

...digital) signal at the end of the reaction (Figure 1). Partitions that did not contain any target DNA at... through homology-directed repair (HDR) (1), or indel mutations resulting from non-homologous end joining...Follow him on Twitter @AnotherLabRat.     References 1. Miyaoka, Y. et al. Isolation of single-base genome-edited...your genome edits, this paper provides detailed instructions on how to design such assays, in addition to...great deal of precision. This means digital PCR provides an absolute measure of target abundance, does ...what kind of assays can be used to sniff out your desired mutations. The assays you will use aren’t that ...often used in real time PCR. These assays can be designed to detect either a donor sequence integrated through...

...BFP2.     Figure 1: CROPseq-multi uses two sgRNAs with internal barcodes (iBARs), multiplexed using...transcript by endogenous processing enzymes (Figure 1). To ensure this tRNA processing only occurs on the...served as positive control (cis-ctrl). v1, vector 1; v2, vector 2; BD, binding domain; SDS, splice donor...)genome editing and gene therapy. Nat Commun., 14(1), 6578. https://doi.org/10.1038/s41467-023-42386-0...separation of the multiplexed elements. The team designed sgRNAs that include an internal barcode and separated... have been made since prime editing was first described in Anzalone et al., 2019, including modifications... sparsely-labeled field of view that was more desirable than having many closely-packed labeled cells....

...include mEGFP, mCherry, and HaloTag. Figure 1: (a) Anti-FLAG frankenbody (FB) construction from ...Comm. 2019. https://doi.org/10.1038/s41467-019-10846-1.   Optimized genetic screening technique using Cas12a... to Cas9’s larger expression requirements (Figure 1), but Cas12a has generally been underutilized in genetic... encoding multiple guides. While two guides require two arrays for Cas9, two guides can be streamlined...applications. This set of new anti-FLAG frankebodies includes mEGFP, mRuby2, iRFP713, SNAP-tag, and HaloTag ...target multiple genes within a single cassette provides increased simplicity for combinatorial genetic...cost-effective alternative to commercial ladders which also provides a useful yet low-pressure teaching opportunity...

...between 2 loxp sites and is excised by Cre (Figure 1). This results in one loxp site and one lox2272 site...cause of this spurious expression?   Figure 1.  Cre-recombinase binds to either pair of lox sites...recombination and can exhibit recombination levels as high as 1-3%. In light of this finding, we now include guidance...Efficiency, Tropism and Axonal Transport of AAV Serotypes 1, 2, 5, 6, 8 and 9 in the Mouse Brain. PLoS ONE 8:e76310...Vectors Pseudotyped with Viral Capsids from Serotypes 1, 2, and 5 Display Differential Efficiency and Cell...experiments to minimize it. Overview of DIO/FLEx vector design Recombinase-dependent vectors utilize pairs of ...between them. Early recombinase-dependent vector designs used a lox-STOP-lox (LSL) or frt-STOP-frt (FSF)...

... and reproducibility of a study’s findings. Table 1: Biased Genotyping Options Technique Sequence Information...sequencing doesn’t tell the whole story Figure 1: Schematic representations of the indicated unbiased...types Must be verified with cell-based method IDLV 1% Cell-based Programmable, can detect DSBs in live ...magazine.scienceforthepeople.org/.     References 1. Fu, Yanfang, et al. "High-frequency off-target mutagenesis...next-generation sequencing." BMC genomics 15.1 (2014): 1. PubMed PMID: 25409780. PubMed Central PMCID: PMC4246457...good starting point for most researchers as it provides a list of putative off-target sites that they ... approach misses larger mismatches (e.g. six nucleotides) that may still lead to off-target double-stranded...

...available. Table 1 compares some general advantages and disadvantages of each approach. Table 1: Properties...alternatives to fluorescent proteins. Figure 1: Firefly, from Wikimedia Commons. The North American...abdomen to produce yellow-green light (Figure 2). Despite the title of this post, this reaction is not fluorescence...fluorescence! Chemiluminescence, which includes all bioluminescence, is the production of light by a chemical...assays (low background, no photobleaching) but provides flexibility by pairing the luciferase with a variety...luciferase output at different wavelengths. Addgene provides empty backbones with the luciferase gene, a variety...

... you’d like to use to generate your new fly line: 1. Where would you like the gene to be incorporated ...control exactly in which locus your transgene ends up (1): Random insertion has the advantage that you can...he still reads “#” as a “pound symbol”. References 1. J. R. Bateman, et al. Site-Specific Transformation...Harvard University. In my previous two posts, I’ve described the fundamentals of how to work with Drosophila...Drosophila as an experimental model organism. I then described the Gal4/UAS system used by geneticists to study....e. there won’t be positioning effects). When designing experiments where you want to express multiple...wondering, “How do I know which of my flies have the desired integration?” Not to worry! In flies, phenotypic...

... experimented with a group mentoring format where 1 mentor met with a group of mentees to get more “bang...The groups in these programs typically consist of 1-2 mentors and 3-5 mentees who meet together for an...get together. If you all work at the same place, a 1-2 hour lunch on a regular schedule might work. If ... for over 10 years. Many successful scientists describe having a “posse” of mentors as one key to their...Ellen Daniell. In “Every Other Thursday,” Daniell describes a group of 7 women scientists who met every other...increases the diversity of input and perspectives Provides a good mechanism for accountability that makes...leadership and communication skills. The leader also provides materials such as pre-reads, videos or exercises...

...narrowed it down to three choices.   Fig. 1: Lin, Abi, or Blottie? Which one suits me best?   ... as they introduce their new friend…  Fig. 1: Meet...well, frankly, we're not sure yet!   This...the final four will receive some rockin’ newly designed Addgene swag. Good luck and happy naming!   ...

... factors such as PAX7, PAX6, AP-2 alpha and ISLET-1. In addition, we distribute monoclonal antibodies ...Monoclonal Antibody Research Institute dedicated to: 1) developing new ways of generating antibodies, for... which results in neutralization or targeted destruction.  Antibodies are composed of two light chains...

...new combinations of PE protein components (Figure 1), an altered epegRNA delivered with an enhanced pU6cm...amplification of cargo, and heat treatment.  Figure 1: Left: recombined prime editing tools PE2max-NC and...conformational dynamics of the motor protein kinesin-1, the Twelvetrees Lab took advantage of the self-labeling..., Craggs, T.D., & Twelvetrees A.E. (2024) Kinesin-1 is highly flexible and adopts an open conformation...Optimized dicot prime editing enables heritable desired edits in tomato and Arabidopsis. Nature Plants,...

...available from their original sources. Figure 1: Biological repositories that distribute mouse strains... from Reproducibility for Everyone. Case study 1: Cell lines Many of the reagents used to investigate...COVID-19: an observational study. The Lancet Microbe 1:e14–e23 . https://doi.org/10.1016/s2666-5247(20)30004...distribution of cell lines from repositories on opposite sides of the world shows that science is truly a global... et al., 2006), the hACE2 transgenic mice were designed after it was discovered that SARS-CoV, which entered...dedicated facilities and manpower that a repository provides. Repositories around the world facilitate the ...

...required for replication and transcription (Figure 1). All viral genes are removed and replaced by a single...cells that stably express T7 (e.g. BSR T7/5 cells) (1). We hope that minigenomes will greatly enhance our...transcription of negative strand RNA viruses.   References 1. Buchholz, Ursula J., Stefan Finke, and Karl-Klaus...reverse genetics. Springer Berlin Heidelberg, 2004. 1-41. PubMed PMID:15298166. 3. Hoenen, Thomas, et al...minigenome system Minigenome systems, such as the one described here, are extremely useful research tools. In ...

...Collection Fluorescent tagging strategy Figure 1: Gene editing is the addition of a useful coding sequence...donor plasmid contains 1 kb homology arms matching the gene of interest, a linker, and the GFP tag sequence...Institute website at alleninstitute.org. References 1. Roberts, Brock, et al. "Systematic gene tagging using... later this year. Designing the donor plasmid Figure 2: Donor plasmid design strategy. A donor ...with each cell line. The editing strategy and design described above can be used to create similar donor ...GFP tag and 1kb of homologous DNA flanking both sides (about 2.7kb total)—is inserted into a plasmid backbone...

...various cyan fluorescent proteins is depicted in figure 1. Of note, the photobleaching rate does not depend ...to be used with the fluorescent protein. In figure 1 we provide an example of such a comparison between...the variants is comparable (lower panel of figure 1). To avoid changes in excitation power, it is important...different ways. We describe a number of issues with some of the experimental designs used to make these...of Amsterdam. The previous post in this series described a practical approach to selecting a bright fluorescent... protein. Photobleaching is the irreversible destruction of a fluorophore under the influence of light...photobleach at some point. For live-cell imaging, it is desirable to have fluorescent proteins that are photostable...

...Cas9 can be used to modify any desired genomic target provided that (1) the sequence is unique compared...will cover three ways to circumvent this limitation: 1) the use of novel S. pyogenes Cas9 variants with varying...Cas9 from Staphylococcus aureus (SaCas9) is about 1 kilobase smaller than SpCas9, so it can be packaged...Biol 19: . https://doi.org/10.1186/s13059-018-1591-1 Cox DBT, Gootenberg JS, Abudayyeh OO, Franklin B, ... may be unintentionally mutated along with the desired target locus. The PAM sequence is of particular...

...neuron which generates an action potential (Figure 1). This light-induced excitation can take place in ...channelrhodopsin activation to a small volume of brain tissue (~1 mm3). Current channelrhodopsins also need high-intensity...fiber-optic cables into the brain. Figure 1: With no blue light, the channelrhodopsin remains ... model to help them construct “designer” channelrhodopsins with desired characteristics. The model was...Viviana Gradinaru’s lab used machine learning to design new channelrhodopsins that are more light sensitive...constrained and poorly understood. It’s hard to design new channelrhodopsins with high-throughput proteins...Using machine-learning to overcome channelrhodopsin design challenges To overcome these challenges, the Arnold...

...standard for genetically-encoded FRET donors (Fig. 1). Scores of FP variants have been created, thereby...with finesse using tRNA suppression technology (Fig. 1) (Dumas, Lercher, Spicer, & Davis, 2015). By using...orthogonal labeling strategies are presented (Fig. 1) which can be carried out using Addgene plasmids. ...involved in skeletal muscle contraction.   References 1. Degorce, François, et al. "HTRF: a technology tailored... transfer measurements of ryanodine receptor type 1 structure using a novel site-specific labeling method...package. One important concept in FRET experimental design is that the targeting specificity requirements ...codon (often amber i.e. UAG) engineered into a desired site. During translation, the novel tRNA/unnatural...

... is conjugated to a fluorophore (Figure 1).   Figure 1: Comparison between how direct immunofluorescence...important considerations are to make sure your secondary 1) will recognize the species of your primary antibody...New York, pp 299–311. https://doi.org/10.1007/978-1-4939-8935-5_26 Stuurman N. (2012) Introduction to ...antibody-based technique, validation and proper experimental design are essential. In this article, we’ll go through...steps include: counterstaining, mounting sample on slides, and confocal   Figure 2: High-level overview...

..., including those found at Addgene! Figure 1: Strategy for assembling the barcoded gRNA library...pool-clone them into the pAWp28 storage vector (Figure 1): Forward oligo: 5’- CACCGNNNNNNNNNNNNNNNNNNNGTTTGGGTCTTCGAGAAGACCTATTCXXXXXXXXC... He can be contacted at [email protected].   References 1. Cheng, Allen A., Huiming Ding, and Timothy K. Lu.... Blog Learn Best Practices for Designing Your gRNA Read How Barcodes Can Be Used in Lineage Tracing Read...genetic perturbations. The ease with which one can design and synthesize CRISPR guide RNAs (gRNAs) for genome...their functions. The continual advancements in gRNA design necessary to achieve maximal on-target and minimal...regulation in biological systems. The CombiGEM platform provides a means to create barcoded gRNA libraries that...

...unleashes its cargo – its own genome.   Fig. 1: Viral particle infection into a host cell. Image ...Vector Resources at Addgene! References and Resources 1. Roe T, et al. “Integration of murine leukemia virus...nuclear import of human immunodeficiency virus type 1 preintegration complexes.” Proc. Natl. Acad. Sci. ...target cells and organisms, and when that cargo is designed to make you sick…well, you feel it. But the same...host genome or temporarily express whatever it encodes. This ability to infect cells with a high efficiency...

...were found on the plasma membrane (top panel in Fig. 1). Of course, this was problematic, because the DREADD...plasma membranes in both species (bottom panel in Fig. 1). Our results indicate that the tag protein may have...protein of interest the membrane.   Figure 1: Electron microscope images showing ultrastructural...protein-coupled receptor, one of the Designer Receptor Exclusively Activated by Designer Drugs (DREADDs) (Armbruster...bottom). The images show examples of dendrites (blue shades) in the monkey amygdala. Red and green arrows point...deciding on the appropriate tag, the experimental design should be the main guidance. The results from our...Raper J, Hu X, Paré J, Bonaventura J, Richie CT, Michaelides M, Mueller SAL, Roseboom PH, Oler JA, Kalin NH...

...Transfer Vectors   Expressing your gene of interest pMKO.1 GFP (Plasmid #10676) Retroviral vector for cloning...GFP expression.  Cloning and expressing RNAi pMKO.1 puro (Plasmid #8452) RNA interference (RNAi) is an...information and a detailed protocol on how to use the pLKO.1 vector can be found here. Cre-Lox technology Puro....of the Retroviridae or Retrovirus family, which includes the gamma-retrovirus and lentivirus genera, have...dividing and non-dividing cells. More detailed descriptions of the lentiviral elements and the 2nd and 3rd...#48201) This retroviral vector from the Gage lab encodes GFP and Cre-recombinase. The Cre-lox system is ...building block Entry vectors and 5 lentiviral Destination vectors. These vectors can induce combinatorial...

...overlap with the negative control population (Figure 1). Based on where the GFP- population ends, you can...total cells present in the gate.   Figure 1: Example of yes-no flow plot of the (negative) control...for a yes-no dot plot, like the one shown in Figure 1.  Figure 3: An example of a histogram plot ... that you know how to read flow plots and have designed your first flow panel, you’ll load your samples...

...vector (abR, Figure 1). The resolution step (“double crossover”) which leads to the desired mutation and excision... requires selection. These two steps are (Figure 1): The initial plasmid integration step (the so-called... loss of a specific phenotype (using sacB, Figure 1, further discussed below).  Counter-selectable marker...Current protocols in molecular biology 106.1 (2014): 1-16. PubMed PMID: 24733238. Additional resources on...scarless mutations, ones that don’t leave any undesired scars in the chromosome. These methods roughly... recombination enzymes can also accomplish the desired genomic modification. This technique is frequently...the pDS132 MCS with a versatile, synthetic MCS (designed with the help of the DNA tuner program) (Latynski...

...interactions with other peptides, like an Fc-binding domain (Figure 1).  Figure 1: Examples of sites...sites on antibodies used for conjugation. 1) Endogenous lysine residues, which can be found throughout the...we would be remiss not to at least mention. Oligonucleotides allow for sensitive detection of low abundance...Strong protein-protein interactions with other peptides. Created with BioRender.com.     Many biologists... conjugation. But hopefully, this introduction provides a good base from which to dive deeper!References...

...succumb to the toxicity of CcdB.   Figure 1: Image from Bernard, P., et al. showing the pKIL 18...submit a guest blog entry of your own! References 1. Bernard, P. “Positive Selection of Recombinant DNA... plasmid ccdB killer gene”. Gene. 1994 Oct 11;148(1):71-4. PubMed PMID: 7926841. 4. Bernard, P., et al... plasmid maintenance during cell division. ccdB codes for the toxic protein (CcdB) that acts as a DNA ...death. ccdA, another gene found in the ccd operon, codes for the antitoxin protein (CcdA) that protects the...of ccdB, leading to positive identification of desired clones.   ...Becomes a powerful cloning tool... efficiently, as those that do not contain the desired insert should not grow.   Figure 2: Image...

...separation is maintained by 2 sets of interactions : 1. Interactions between the non-target strand and a ...theoretically decrease off-target cutting. Figure 1: Strand separation and target DNA binding to the Cas9... improvements in genome engineering! References 1. Slaymaker, Ian M., et al. "Rationally engineered ... nuclease activity of the Cas9 nuclease itself. Design and testing of eSpCas9 Looking at the structure...and the negatively charged DNA and therefore destabilize basal strand separation. This decreased strand... choice and get improved on-target specificity. Design and testing of SpCas9-HF1 Kleinstiver et al and...Joining Repairs Double Strand Breaks Get Tips on Designing Your gRNA Browse Our CRISPR 101 Series Resources...

...the other (Sandomenico et al., 2020). Figure 1: Comparison between the IgG antibody and scFv. ...intensity of a tagged protein. SunTag has two components: 1) a protein of interest that’s tagged with 10-24 copies...Application. Clinical and Developmental Immunology 2012:1–15 . https://doi.org/10.1155/2012/980250  Bates A,...without disulfide bonds, made by molecular evolution 1 1Edited by I. A. Wilson. Journal of Molecular Biology...recombinant antibody. They are ~25 kDa single polypeptides that contain the variable light chain (VL) and...lethal to animals.  In the clinic, scFvs’ size also provides advantages over antibodies (Ahmed et al., 2012... bond between the VL and VH domains. This bond provides stability and solubility for the scFv (Gaciarz...

... employing HDR. Ma et al. moved forward with gRNA 1 to conduct microinjection experiments in S phase zygotes...instead from the mother’s wild type allele (Figure 1). 72.4% of embryos carried two wild-type copies of...proceeded through NHEJ rather than HDR. Figure 1: Homology-directed repair in targeted zygotes proceeds...donated skin, blood and semen samples. Ma et al. designed plasmids containing Cas9 and two different gRNAs... at 3 days post fertilization. Since Ma et al. designed a repair template with two synonymous mutations...thus quieting some fears about the creation of “designer babies.” The differences in editing between iPS...

...difficult-to-manipulate primary cells, achieving knockdown within 1-2 hr of reagent delivery. TRIM-Away is suitable for... inducer levels. That is, if transcription factor 1 controls expression of transcription factor 2, which...modulating the induction of transcription factors 1 and 2. See Aranda-Díaz et al. for more information...TRIM21 recruits ubiquitin and the proteasome to destroy antibody-bound pathogens. Clift et al. found that...necessary for studying endosomes, the Hell lab designed their super-resolution pH indicators (SRpHi) biosensors...sgRNAs are used with FIRE-Cas9, experiments can be designed to target multiple loci at once. In addition, ...

...want a low affinity calcium biosensor, so that the (1) binding of calcium, (2) allosteric rearrangements... to a red fluorescent state only by simultaneous (1) user-controlled illumination with violet light and...fluorescent indicators. Nature protocols. 2006 Aug;1(3):1057-65. Resources on Addgene.org Addgene’s AAV... There are several calcium reporters specially designed for the aims and requirements of in vivo studies...populations of interest. Many of the calcium sensors described are also already packaged into viral vectors and... L, Campbell RE. Structure-and mechanism-guided design of single fluorescent protein-based biosensors....

... the Roska lab used four different design strategies: Strategy #1 (ProA): Create promoters with sequences...cell types in mice. Figure 1: The four strategies used to design synthetic promoters. Legend: TSS...rate of targeted expression. Surprisingly, <1% of synthetic promoters replicated the expression specificity...expression in the retina.  Four approaches for designing synthetic promoters  To build a library of synthetic...promoters with the help of epigenetic analysis and designed using stretches of DNA that were transcriptionally...class of cells  Of the four different promoter designs, promoters from strategy #4 had the highest rate...here.  Applications for synthetic promoter AAVs Besides restricting AAV transgene expression to particular...

... : Niroshi and Ben describe their projects 0:28 - 1:12 : Introduction from Tyler 1:13 - 2:02 : Niroshi...

...SpCas9, is NGG. Assuming all nucleotides are represented equally, that’s only a 1/16 chance of having a PAM... NYN/NRN NYN is less effective than NRN Table 1 - Summary of PAM-flexible Cas enzymes Are we truly...editable with CRISPR technology.     Fig. 1: SpCas9 has sparse PAM coverage in the human genome. ...editing landscape. The need for PAM-less editing   Guides for CRISPR are typically selected by the ‘quality...sequence is suitable, but there is no PAM next to the desired cut site, then a further-away gRNA where there ... may wonder: how close is close enough for the desired edit to still occur if the cut site is sub-optimal...it depends, but generally the efficiency of the desired edit is reduced by over 50% when the cut site is...

...Zhang et al., 2015). In this system a modified TIR-1 protein can degrade degron-tagged proteins in an auxin...splitting cGAL4 in two and binding each half to a gp41-1-N-intein. Split inteins are protein domains that naturally...mutations with unc- 119 + selection." MicroPublication 1 (2018): 2–3. Wang, Han, et al. "cGAL, a temperature-robust...of 3’UTRs, the Arizona State University (ASU) Biodesign institute developed a 3’UTR database specifically...specifically for C. elegans. This database provides detailed information about C. elegans 3’UTRs structures, ...imaging processing. The combination of these methods provides unique opportunities to develop computational ...Addgene In addition to the wealth of open resources described above, there were many plasmid tools presented...