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... The three VP proteins assemble into capsid at a 1:1:10 ratio (VP1:VP2:VP3). There are eleven naturally... Figure 1. The RepCap plasmid and its products. Created with BioRender.com   Replication (rep) gene...Rep78/68 play a key role in AAV genome replication. Replication initiates from the AAV genome ITRs on ...contains the proteins needed for the virus to replicate,  and the transfer plasmid which contains the ...aka the RepCap plasmid encodes two genes: the replication (rep) gene and the capsid (cap) gene. In wild-type... proteins that are required for viral genome replication and packaging: Rep78, Rep68, Rep52, and Rep40... generate a 3’ hydroxyl group needed for DNA replication, Rep78/68 binds the Rep binding element (RBE)...

...discovered such as Kaede and PS-CFP2 (see Table 1). Table 1: Properties of Selected Irreversible Photoactivatable...) by mutating Threonine 203 to Histidine (T203H) (1). wtGFP normally contains a mixed population of neutral...in fluorescence when illuminated with blue light (1). Similar to PA-GFP, scientists later developed red...fixation conditions to preserve cellular structures (0.5-1% osmium tetroxide). These conditions destroy most ...with extraordinary molecular detail.   References 1.Patterson, George H., and Jennifer Lippincott-Schwartz...is believing." Annu. Rev. Biomed. Eng. 10 (2008): 1-38. PubMed PMID:18647110. Additional Resources on ...up. Applications of photoactivatable fluorescent proteins Some of the most important applications for ...

...that the culprit for these unusual cases was either 1) a physiological effect of SARS-CoV-2 toward destabilizing...an AdV transfer plasmid with the E1 and E3 (Early 1 and Early 3) genes deleted. These genes’ functions...certain contexts (Bauzon et al., 2003).        Figure 1: The adenovirus life cycle. Image reused from Kremer... review of the evidence. Emerg Microbes Infect, 8(1), 1679–1687. https://doi.org/10.1080/22221751.2019.1690953...574–580. https://doi.org/10.1038/s41586-023-05949-1. PMID: 36996871. Tatsis, N., Fitzgerald, J. C., Reyes-Sandoval...and late gene transcription) is demarcated by viral genome replication (Harrach, 2014). Given that the...encapsidate new virions) are not expressed. Note that replication-competent AdV vectors also exist and are used...

...origin of replication (p15A) worked best under a wide range of inducer concentrations (0.1-1% L-arabinose...bacteria have colonized (Fig. 1c).     Fig. 1: Expressing bARGSer in E. coli. (a) Plasmid diagram...ultrasound contrast. In all the cell lines we tested, 1 µg/mL doxycycline was sufficient to saturate expression...engineering a cell line, cotransfect the mARG cassettes 2:1 (A:N-V) with PiggyBac transposase and outgrow the ... tumors and tumor-homing bacteria. Nat Biotechnol 1–13 (2023) doi:10.1038/s41587-022-01581-y  ... they are based on light which is very easily scattered by tissue. Ultrasound, on the other hand, is a...protein nanostructures called gas vesicles, which scatter sound waves and thereby produce ultrasound contrast...

.... Level 1: Assembly of multiple Level 0 parts into TUs. Level 2: Assembly of multiple Level 1 TUs into...: promoter, gene, and terminator.     Fig 1. MoClo enables individual parts to be assembled into... peptide can easily be tested by generating Level 1 TUs with unique promoters, and the ideal phosphatase...most obvious example of an individual part, this category also includes promoters, UTRs, terminators, etc...multi-purpose it can be. Let’s review a list of MoClo applications. Genetic circuits Most things in biology are...performing production and subsequent necessary modification of these chemical products. The flexible nature...genotype and phenotype is best described with “it’s complicated.” Applying MoClo Interested in using MoClo? You...

...much more favorable than previous methods yielding 1-10% knock-in. Read on to learn how you can make CRISPR...one NHEJ-repaired indel, often occur. Figure 1: In conventional gene targeting (A), you must first... drug induction systems (ttA or rtTA) are between 1 to 2 kb in length. There are plenty of Addgene plasmid...CRISPR ribonucleoproteins.” Genome Biol. 18 (2017) (1):92. DOI: 10.1186/s13059-017-1220-4 PubMed PMID: 28511701... using long ssDNA donors.” Nat Protoc. 13 (2018) (1):195-215. DOI: 10.1038/nprot.2017.153 PubMed PMID:...engineering using CRISPR nucleases. Genome Biol. 19 (2018) (1):25. DOI: 10.1186/s13059-018-1400-x PubMed PMID: 29482575... allele To design a knock-in allele at a given location, you’ll need to design only a single guide and...

...2008) Transposons: The jumping genes. Nature Education 1(1):204. https://www.nature.com/scitable/topicpage... They transpose by a ‘copy-paste’ mechanism (Fig. 1). They first copy themselves as RNA transcripts and...their host’s genome.   There are two types of Class 1 TEs: those with long terminal repeats (LTRs) and those...reverse transcriptase activities.    Figure 1: Overview of retrotransposon transposition. Retrotransposons...CRISPR-Cas system. This system has four major components: 1) a CRISPR RNA (crRNA), 2) four proteins (TniQ, Cas8... It generally stays put, which makes it easy to locate in a genome. But there is a type of mobile DNA,... hyperactive and likes to jump around from one location in the genome to another. This jumping is what...

...harness non-homologous end joining repair (NHEJ) [1]. Our initial work was with TALENs, in collaboration...from the University of Pennsylvania.     References: 1. Lee HJ, Kim E, Kim JS. Targeted chromosomal deletions...using zinc finger nucleases. Genome Res. 2010 Jan;20(1):81-89. PubMed. 2. Bauer DE, Kamran SC, Lessard S,... of Visualized Experiments (JOVE). The video publication by Stuart Orkin and Daniel Bauer's labs details... size and frequency [3]. There are several applications in which the production of deletions by paired...useful to study haploinsufficiency. Subsequent modifications at the remaining allele within the deletion ...

...translation, or chromatin modifications of complementary gene sequences (Figure 1). As a result, gene silencing...benefited from the ease of use in dsRNA applications. Figure 1: Schematic of an RNAi-inducing transgene...dsRNA via an RNase III enzyme named Dicer (Figure 1). The plant kingdom has expanded its repertoire of...in the biogenesis of different small RNAs (Figure 1). These siRNAs can exert their gene silencing effect...complementary to your gene target as inverted repeats (Figure 1). With this technique, multiple genes can be simultaneously...selection of plants undergoing RNAi, and LIIbeta F 1-2 RNAi, which allows assembly of intron-spliced hpRNAs...silencing can be easily induced by exogenous application of long or short dsRNA. However, it is important...

... different steps and materials required for each [1, 2]. This protocol [3] provides, in exquisite detail...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 ...There are several important considerations with fabrication of the fiber optics needed for optogenetics. ... for the implant depends on the dorsal/ventral location of your region of stimulation (the z-axis bregma...emitted from the end of the fiber optic implant scatters a great deal once it enters tissue, placement ...

...’s fused to the C-terminus of DsRed.T3. See Table 1 for a summary of Rosella’s excitation and emission...fluorescence Rosella emits based on its localization. Table 1: Summary of Rosella’s excitation and emission spectra... variants are available from Addgene. References 1. Rosado, C., Mijaljica, D., Hatzinisiriou, I., Prescott...single fluorescent biosensor. Scientific Reports,5(1). doi:10.1038/srep12397. PubMed PMID: 26215030. PubMed...radioactivity in the culture supernatant (this is indicative of protein degradation). To control for protein...studying autophagy. This method relies on the identification of autophagic structures based on morphology...

...versatility of the basic antibody structure (Figure 1). Antibodies can be broken up into smaller parts, ...antibodies for a specific purpose. Figure 1: Overview of antibody engineering methods. (A) Basic...,"rgb":"rgb(0, 82, 169)","rgba":"rgba(0, 82, 169, 1)"},"button_font":{"size":19,"styles":{"font-family...:"rgb(255, 255, 255)","rgba":"rgba(255, 255, 255, 1)"}}{% endraw %}{% end_module_attribute %}{% module_attribute...Molecular Biology, 29–62. https://doi.org/10.1007/978-1-0716-2285-8_2 McMahon, C., Baier, A. S., Pascolutti...by PCR. Current Protocols in Molecular Biology, 51(1). https://doi.org/10.1002/0471142727.mb0803s51 Additional...equipment on a lab bench. The best part? Less complicated math. Bioengineering is a field that has been...

...118). Humana Press. https://doi.org/10.1385/1-59259-301-1:99 Resources on Addgene.org Addgene antibodies...antibody for that!   Fig. 1 - Diagram of His-tagged protein purification work flow by nickel column....cleaved after purification, so as not to perturb protein function for downstream applications. Tag cleavage...thoughtful planning, affinity tags can make protein purification a cinch. Types of affinity tags, the proteins... Affinity tags are used primarily as protein purification tools (and can have secondary functions as detection...single protein is expressed, with the intent of purification, it will be expressed along with all other cellular...available antibodies, resins, beads, and other purification aids readily available. Choosing the best tag...

...requestors. We also offer export license applications for VSV and SARS-COV-1 plasmids. Export control license...  We have offered VSV and SARS-COV-1 plasmids under this application process since May 2020. We find this...excited to announce we now offer an export license application for rabies materials! Some of the materials in...export-controlled materials in the USA, where Addgene is located.  The export license, if granted, allows us to ... applied to cover processing costs. Once the application is approved, Addgene then ships the material ...whenever possible, as we cannot expedite the application process.  More information about export control...

...following four components into 293T cells. Figure 1: The key components of Nanoblades. Image from Mangeot...protein. A single Nanoblade particle can be loaded with 1-4 different gRNAs. Viral envelope proteins To alter... between ~50-70% for these four cell types (Table 1). Additionally, hiPSCs and mouse BM cells still behaved...hepatocytes  Myd88  ~50% Human HSCs  Myd88  ~50% Table 1: Nanoblades editing efficiencies of primary cells....including RNPs for indel formation via NHEJ, precise modification with Homology Directed Repair (HDR), CRISPR ...potential to be used for other types of CRISPR applications. Let’s take a look at the key components of ... which means they are non-infectious and non-replicative. Nanoblades are produced by transfecting the ...

...spectral overlap, the fluorophores must be located within 1-10nm of each other and be oriented appropriately... to make the decision easier. 1. Müller, Sara M., et al. "Quantification of Förster resonance energy transfer...acceptor fluorophore. The typical steps in FRET are: 1. Donor fluorophore excitation by absorption of a photon...diseases and even assist in drug discovery. References 1. Baird, Geoffrey S., et al. "Circular permutation ...have enhanced the ability of labs to answer sophisticated questions about cellular processes. Currently...PMC18018. 2. Miyawaki, Atsushi, et al. "Fluorescent indicators for Ca2+ based on green fluorescent proteins ...

...stimuli. Cre recombinase catalyzes the recombination of DNA sequences located between two loxP sites. ...in research can fall under one of two broad classifications: inbred or outbred. Inbred strains share a ...Knockout mice Knockout mice have undergone genetic modification that alters or eliminates the expression of ...can be used to invert a segment of DNA or to translocate a piece of DNA from one region of the genome ...

...let’s go over the details step-by-step. 1. Digestion Set up restriction digests for your insert (or donor...recipient plasmid : insert ratio” of approximately 1:3. Since the number of base pairs for each varies,...cells. For most standard cloning, you can transform 1-2μl of your ligation reaction into competent cells...at minimum, two transformations after a ligation: 1. Control Transformation containing the ligation mixture...result in few colonies on both plates (unsuccessful 1), in a vector alone plate with many more colonies ...them. An easy way to do this is gel purification. In gel purification, you use a voltage difference across...backbone. Sample results indicative of successful and unsuccessful ligations are indicated below. A successful...

...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 ...calcium indicator (Shemetov et al., 2021). In addition to FRET, intensiometric calcium indicators NIR-GECOs...characteristics, performance, and applications of near-infrared calcium indicators in this recent review (Shcherbakova...information on NIR FPs, their properties and applications. At the end, there is a Table that summarizes...

...In the case of HIV-1 and SARS-CoV-2 detection, AIOD-CRISPR without preamplification was able to detect... to other technologies like CRISPR. Figure 1: An overview of the general schematic of CRISPR/Cas... can be detected – in the zeptomolar range Time: ~1 hour Equipment required: 37°C water bath and 42°C ... Not mentioned in the paper, likely approximately 1-2 hours Equipment needed: Mini PCR machines by DIY-Bio...femtomolar  Time: Not mentioned, likely ~1-2 hours Equipment needed: PCR machines/Heat blocks, Lateral...recombinase polymerase amplification (RPA) using a commercially available amplification kit. In this case,... RNA is extracted and subject to isothermal amplification by primers that target the E and the N2 gene...