...Cambridge office – previously at 1 Kendall Square, but newly relocated to 75 Sidney Street. Then the plasmids...here at Addgene – 500,000 plasmids shipped! That's a 1/2 million plasmids sent to happy scientists around...Have a question? Email: [email protected] Call: +1 (617) 225-9000         ... thank you. 500,000x Thank You! Addgene's new location: 75 Sidney St, Suite 550A, Cambridge, MA 02139...

...type of choice in vitro is known as reprogramming [1]. The process can be divided into two stages: Dedifferentiation...//www.linkedin.com/in/kmukherjeephd/.  References 1. Hochedlinger, K. and R. Jaenisch, Nuclear reprogramming...from human peripheral blood. Cell Stem Cell, 2010. 7(1): p. 15-9. PubMed PMID: 20621044. PubMed Central PMCID...induced pluripotent stem cells. Hepatology, 2010. 51(1): p. 297-305. PubMed PMID: 19998274. PubMed Central... pluripotent stem cells. Nat Biotechnol, 2014. 32(1): p. 84-91. PubMed PMID: 24291815. PubMed Central ...keratinocyte lineage. Methods Mol Biol, 2014. 1195: p. 1-12. PubMed PMID: 24510784. PubMed Central PMCID: PMC4096605...induced pluripotent stem cells. PLoS One, 2010. 5(1): p. e8763. PubMed PMID: 20098701. 31. Tang, Z.H.,...

...disadvantages: Table 1 (Joglekar et al., 2017)  Table 1 (Gutierrez-Guerrero et al., 2020) Table 1 (Cronin et al...polyA tail.   Figure 1: Lentiviral production uses three plasmids: (1) The transfer plasmid, (2)... specific purposes as described previously (Table 1, Gutierrez-Guerrero, et al, 2020). Because retrovirus...The glycoprotein will be incorporated into the replicating rabies virus envelope, travel retrogradely, and... Pseudotypes: Precious Tools to Improve Gene Modification of Hematopoietic Cells for Research and Gene...

...individually or as a complete kit from Addgene. References 1. Goulas, Theodoros, et al. "The pCri system: a vector...protein purification. For a more thorough introduction to pCri, please see the original publication cited...expression and purification: a comprehensive review of affinity tags and microbial applications." Biotechnology... Protein purification can be one of the most stressful lab activities. Working with proteins requires...interest can save a lot of time later. Protein purification specialists recommend doing some research, notably...Gomis-Rüth, aims to take the guesswork out of protein purification through simple and efficient multiplexing. pCri...sites should one need to remove the tag post-purification. A sample of pCri constructs: pCri-1a, -4a ...

...into more specialized cells with specific functions [1]. In many tissues, stem cells serve to replenish/replace...damaged cells that no longer function adequately [1]. Stem cells’ ability to differentiate into multiple...://www.linkedin.com/in/kmukherjeephd/. References 1. Dittrich, R., M.W. Beckmann, and W. Wurfel, Non-embryo-destructive...The potential of muscle stem cells. Dev Cell, 2001. 1(3): p. 333-42. PubMed PMID: 11702945. 6. Cowan, C....preimplantation genetic diagnosis. Hum Reprod, 2008. 23(1): p. 46-53. PubMed PMID: 17989069. 8. Takahashi, K...stem cells are: Their ability to self-renew or replicate while maintaining the undifferentiated “stem” ...Unipotency: As the name suggests, cells under this category can differentiate into only one cell type. Muscle...

...Cells? Yes Yes Longevity in Serum?* No Yes Table 1. Comparing SpCas9 and GeoCas9. *For in vitro Cas9 ...GeoCas9 is active at high temperatures Figure 1: Thermostability of SpCas9 vs GeoCas9. SpCas9 or GeoCas9...from 25-80 °C. Activity is expressed as Kcleave (min-1). SpCas9 has maximum activity from 35-45 °C, while...outperformed SpCas9 at high temperatures. See Figure 1 for a summary of the experimental results. GeoCas9...could also be used with in vitro molecular bio applications that require high temperature reaction conditions...

...the genome, which seem resistant to Cas9 cleavage (1). Both the CRISPR-Cas9 and NgAgo genome engineering...it was first published in Nat Biotech in May 2016 (1), NgAgo has received much attention and the plasmid...Pooran's NgAgo Survey Survey results As of August 1, 2016, a total of 165 researchers responded to the...the cell, the same method used by Gao et al, 2016 (1). View the Complete and Up-to-Date Results of Pooran's... dreams of a sunny day in Edinburgh.   References 1. Gao, Feng, et al. "DNA-guided genome editing using...been excitedly testing NgAgo's genome editing applications. So, how does NgAgo stack up thus far? A few...survey. When asked if they could detect indels (an indication of gene disruption ability) with NgAgo, one respondent...

...premature chain termination 1-10 ug/mL Zeocin Sh bla HeLa, NIH3T3, CHO, COS-1, 293HEK, Jurkat T cells Complexes...effective replication. Cell lines expressing the Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA1) or...Concentation*** Blasticidin bsd  HeLa, NIH3T3, CHO, COS-1, 293HEK Inhibits termination step of translation ... a bacterial origin of replication (ORI) will not allow for plasmid replication in mammalian cells, for... their bacterial counterparts, including how replication occurs and whether selection is necessary for...the nuclear genome, in the absence of plasmid replication, the foreign DNA will be degraded or diluted ...always mission critical to make sure the plasmid replicates in the cell, as many of these experiments are...

...them with TAA (7). This modification allowed the deletion of release factor 1, the protein responsible...cellular proteins are modified through phosphorylation (1). However, the expansion of studies on protein phosphorylation...three components required for translation (Figure 1): A tRNA that recognizes a specific ‘open’ codon ... open reading frame (ORF) of interest (Figure 2). 1. The phosphoserine OTS The backbone of the phosphoserine...Rinehart lab: pCRT7 NT TOPO E17TAG GFP and pGEX-6P-1. The pCRT7 NT TOPO vector contains a mutant pSer-containing... can follow her on Twitter @nieminm.   References 1. Cohen, Philip. "The origins of protein phosphorylation...most inclusive databases of post-translational modifications, identifies a staggering ~250,000 phosphorylation...

...them to target particular cell types. Figure 1: Diagram of AAV components. Created with BioRender.com...depends on your choice of promoter/enhancer. Table 1: AAV capsids and receptors. Capsid Primary receptor...required)2 Human fibroblast growth factor receptor 1 (FGFR1)3 αVβ5 and α5β1 integrins3 Hepatocyte growth...structure of AAV2, so they are formally called rAAV2/1, rAAV2/2, and so on, although we often use abbreviated...efficiency, tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. PloS One, 8(... N.J.), 807, 141–157. https://doi.org/10.1007/978-1-61779-370-7_6 Tabebordbar, M., Lagerborg, K. A., Stanton... and Transduction by Adeno-Associated Virus Types 1 and 6. Journal of Virology, 80(18), 9093–9103. https...

...transformation (Figure 1). As this approach requires no special reagents or purification kits, it is low cost...with linearized vector backbone. DpnI digestion - 1 μl DpnI is added to the PCR mix to remove parental...confirm primer design.  Use minimal template DNA Use 1 ng of PCR template DNA to minimize false positive ... by PCR, so we usually add DMSO (3%) and Betaine (1 M) to the PCR mix. When PCR is not possible Some plasmids...Twitter @jakefwatson and @JGarciaNafria.  References 1. Jones, Douglas H., and Bruce H. Howard. "A rapid ...3’ end binds template DNA for PCR amplification, while modifications and homologous sequences are encoded...multiple modifications A key feature of IVA cloning is the ability to combine multiple modifications. Any ...

...guide for a refresher on CRISPR/Cas9.  Figure 1: Cpf1 is a single RNA-guided endonuclease of a class... than the multi-subunit complex employed by class 1 systems. A putative new class 2 nuclease, Cpf1 (CRISPR...that Cpf1 can tolerate mismatches at gRNA positions 1, 8, 9, and 19-23. Accordingly, the 3’ end of the gRNA...Most gRNAs directed low-frequency Cpf1 cleavage at 1-12 off-target sites; in contrast, SpCas9 may cleave...the diversity of CRISPR-based systems. References 1. Zetsche, Bernd, et al. "Cpf1 is a single RNA-guided...mismatches on modification by AsCpf1 (red) or LbCpf1 (blue). Mismatch positions are indicated below each ... this publication at Addgene. Makarova, Kira S., et al. "An updated evolutionary classification of CRISPR-Cas...

... Table 1: Key differences between original CAPTURE and CAPTURE 2.0 methods.   Applications of CAPTURE...by PCR or next-generation sequencing. Figure 1: Overview of the key steps of the CAPTURE method. ...ligases Expression system Stable cell line expressing 1) FLAG-biotin-tagged dCas9, 2) BirA, and 3) one or ...or more gRNAs Two separate lentiviruses delivering 1) a BioTAP-tagged dCas9 and an eGFP tag, and 2) gRNAs...The Xu lab presented three ways to use CAPTURE 2.0: 1. Multiplex capture of chromatin interactions The Xu... gene’s transcription. Sometimes enhancers are located thousands of base pairs away from the gene they...created a dCas9-based CAPTURE (CRISPR Affinity Purification in situ of Regulatory Elements) method. The ...

...the sample (by less than 1%) and recovered after the magnet was removed (Figure 1). At first, Andrew and...since the initial report in 2023.)     Figure 1: Fluorescence intensity of EGFP-FlavinTag in E. coli...that could be expressed and purified. Although a ~1% change in signal might not make a very powerful tool...Hayward et al. 2024 under CC-BY license.   Video 1: Fluorescence movie of E. coli expressing MagLOV while... tools for familiar applications. But how does one invent a tool for applications that don’t even exist...phenomenon, improve the tool, and develop new applications for magnetoresponsive proteins. Motivated by...organisms) are opaque, which makes most biomedical applications of these tools difficult or impossible. But ...

...Subscribe to CRISPR Blog Posts Applications of Cas13a   Figure 1: Using Cas13a as a diagnostic ...C2c1, C2c2 and C2c3 (short for Class 2, candidate 1, 2, or 3). C2c1 and C2c3 are related to Cpf1, but ...lacks the ability to cleave the RNA target.  Table 1: Comparison on Common CRISPR Enzymes Name  Enzymatic... This system, dubbed SHERLOCK (depicted in figure 1) has been used to differentiate strains of Zika virus...contributed to the update of this post. References 1. Abudayyeh, Omar O., et al. "C2c2 is a single-component...use, functional in most species, and has many applications. That said, SpCas9 is not the only game in town... makes this molecule unique, and the various applications of Cas13a. The origins of Cas13a: An RNA cleaving...

...directed base excision repair proteins. Nature Communications, 12(1), 1384. https://doi.org/10.1038/s41467-021...predictable by machine learning methods. Nature Communications, 12(1), 4902. https://doi.org/10.1038/s41467-021...for more details on how they work!) Figure 1: Base transitions are edits between the purine bases...R-loop structure. In order to bind DNA, Cas9 requires (1) a guide RNA, (2) a complementary DNA sequence, and...given the pithier name CGBE1, for “C → G Base Editor 1,” and had a C → G editing efficiency of up to 68%... the CGBE: DNA polymerase D2 (POLD2), exonuclease 1 (EXO1), and RNA binding motif protein X-linked (RBMX...transversions in human cells. Nature Biotechnology, 39(1), 41–46. https://doi.org/10.1038/s41587-020-0609-x...

...animal, those B cells are fused to myeloma cells at a 1:1 ratio and then cultured in a special medium called...antibodies that recognize proteins. Figure 1: Steps in the generation of hybridomas for monoclonal...Washington (DC): National Academies Press (US); 1999. 1, Generation of Hybridomas: Permanent Cell Lines Secreting...cells and antibody-producing B cells that cannot replicate, meaning that only hybridomas will survive this... Antibodies: Distinguishing Characteristics, Applications, and Information Resources. ILAR Journal 46:... the Addgene blog Learn more about research applications for antibodies in Intro to Antibodies Read about...

...that have now become hallmarks of ideal FP timers: 1. Ratiometric determination of expression time The ...the FT series matures from blue to red (see Table 1 for emission and absorption spectra). Subach et al...times and varied blue-red maturation times (Table 1). These separate proteins should be useful for monitoring...cellular events occurring at varied time scales.  Table 1: Monomeric FP Timers and Associated Plasmids from ... know in the comments section below! References 1. Terskikh, Alexey, et al. ""Fluorescent timer": protein... (green or blue) within a population of timer indicates the age of the pool: the higher the ratio, the...-FT-N1 pTRE-Slow-FT pBAD/HisB-Slow-FT -  Applications of fluorescent protein timers FP timers have...

...evaluate all of the pegRNAs in one experiment (Figure 1). Through this method, we found key determinants of...diverse edit types and cell contexts. Figure 1: High-throughput screening of pegRNA efficiency and...Article 3. https://doi.org/10.1038/s41576-022-00541-1 Kim, H. K., Kim, Y., Lee, S., Min, S., Bae, J. Y.,...DNA repair determinants. Nature Biotechnology 2023, 1–11. https://doi.org/10.1038/s41587-023-01678-y Li,...editing systems in multiple cell types. Cell, 186, 1–17. https://doi.org/10.1016/j.cell.2023.03.034 Resources...genome editing technology that allows precise modifications of DNA (replacements, small insertions, and ...configurations can be used to introduce specific target modifications. Since this means we could design hundreds to...

... arms. Nat Chem Biol., 308(20): 1-9 (2020). 10.1038/s41589-019-0432-1 Mehdi Banan. Recent advances in ...before attempting knock-ins by HDR.   Fig. 1: Early steps in repair of a DNA double strand break...traditionally have larger homology arms in the 500 bp to 1 kb range, however, shorter homology tracts have recently...breaks. J Biol Chem., 293(27): 10524-10535 (2018). 1 10.1074/jbc.TM118.000372 Yang, H., Ren, S., Yu, S....cut site location Position your CRISPR cut site as close as possible to the knock-in location. HDR depends...sequence to be incorporated at a precise genomic location, with little to no room for error. It’s no surprise...nutrients in their media, they will not undergo DNA replication, so make sure you are keeping an eye on those...

...transcription activation system co-opted from yeast (1). It is a Drosophila geneticist’s main workhorse to...he still reads “#” as a “pound symbol”. References 1. A. H. Brand, N. Perrimon, Targeted gene expression... A Fly Geneticist’s Swiss Army Knife, Genesis 34, 1–15 (2002).PubMed PMID: 12324939. 3. S. E. McGuire,...Versatile Toolkit for Gene Expression in Drosophila, 3, 1–10 (2015). PubMed PMID: 21356876. 5. J. Chow, Z. Marka...as overexpression of each gene individually. Applications of the Gal4/UAS system There are a number of...lines. In the meantime, be sure to browse the publication references and web links from my first post and...

...incubation of 1° and 2° Must use different species and/or different IgG subclasses of 1° for multi-color...displacement 2° incubation can be skipped by pre-binding to 1° Monovalent Monoclonal Only anti- rabbit and -...dyes are also commercially available. References 1. Pleiner, T., Bates, M., & Görlich, D. (2017). A toolbox...NIH’s Developmental Studies Hybridoma Bank (DSHB). 1 ml of antibody-containing hybridoma supernatant is...in bacteria and extracted with common protein purification methods. Read on to learn more about nanobodies..., Alexa Fluor 568, or Alexa Fluor 647 before application to the sample. Staining with this multi-color...PubMed PMID: 29263082 Find plasmids from this publication at Addgene. 2. Bates, M., Böhning, M.I., Chug...

...origin of replication (oriP) element and the cis-acting EBNA-1 from Epstein-Barr virus. EBNA-1 binds oriP... be completely removed.  Replicating oriP/Epstein-Barr nuclear antigen-1 (EBNA1) based episomes: These...reprogramming method Most iPSC research falls into two categories: 1) studies focused on better understanding the...Difficult to purify reprogramming proteins. Table 1: Key Features of Different Methods for Generating ...plasmid-based expression requires serial transfection of 1 or 2 plasmids that express the reprogramming factors ...iPSCs experiences in the comments below! References  1. Sano, Masayuki, Minoru Iijima, Manami Ohtaka, and...Methods figures were adapted from this reference. 1. Malik, Nasir, and Mahendra S. Rao. "A Review of the...

...sighted readers.    Figure 1: An image from AAV Purification by Iodixanol Gradient Ultracentrifugation... it? The image in Figure 1 appears on our protocol page for AAV Purification by Iodixanol Gradient Ultracentrifugation...intended.)  Steps to clear writing Let’s use Figure 1 as our example. Remember, our goal is to use words... share with the rest of the workshop.  For Figure 1, Addgene chose this alt text: "The left panel is a...principles we developed to use in our website and educational content conflicted with plain language guidelines...Stupid, overwhelmed, discouraged, tired…words that indicated there were many barriers between our audience ...

...production of the pro-inflammatory cytokine interleukin IL-1 beta in the dorsal raphe nucleus (DRN), a region thought... a transgenic mouse harbouring a Cre-dependent IL-1 beta overexpression cassette. Expression of Cre in...in these neurons turns on overexpression of IL-1 beta. They tested the mice in a series of behavioral ...risky), or a closed arm (safe). Mice with higher IL-1 beta production spent more time in the open arm and... part of the apparatus, it turned out that the IL-1 beta overexpressing mice spent more time in the risky...have made great strides to map brain regions and allocate specific functions to them. Electrical recordings...long-lasting antidepressant effects." Nature communications 10.1 (2019): 223. PubMed PMID: 30644390. PubMed...

...such as IL-8, IFNγ, and colony stimulating factor 1 (CSF-1). Additionally, tumor cells recruit regulatory...introduced the “Three Es of Immunoediting” nomenclature. 1. Eliminate - Latent tumors are recognized by effector...the tumor, activates hypoxia inducing factor (HIF-1), which promotes the production of VEGF to increase...This study used CRISPR gene editing to knockout PD-1 on immune cells harvested from patients and thereby...exploring new avenues in healthcare.   References 1. Vinay, Dass S., et al. "Immune evasion in cancer:...lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial." The Lancet 385.9967 (2015)...immune response to the development of tumors is a complicated and dynamic phenomenon that is interestingly ...

... all replicate in E. coli, which is the most commonly used bacteria in lab manipulations. Table 1 has ...https://doi.org/10.1007/978-1-61779-197-0_19  Meyer, R. (2009). Replication and conjugative mobilization...reduced metabolic load (Meyer 2009). Figure 1: Idealized examples of a narrow host range plasmid...species these plasmids are compatible with.  Table 1: Non-exhaustive list of common broad host range plasmids...for DNA cloning in Gram-negative bacteria. Gene, 70(1), 191–197. https://doi.org/10.1016/0378-1119(88)90117...ori2, ori3) and replication initiation proteins (RepA, RepB) necessary for replication in various hosts...the ori and allow for replication. However, if plasmids have their own replication machinery such as Rep...

... the example shown in Figure 1, injected retrograde AAVs at one location result in opsin expression in...from the paper for designing experiments with AAV: 1. Getting your viral vector into your cell of interest... administration routes for neuroscience see Table 1 from the paper. 2. Manipulate your cell of interest...experiments! Late last year, we hosted our first AAV Education and Development workshop aimed at brainstorming...even further. For example, injecting AAVs at one location can result in expression of an optogenetic payload...

...choosing direction of a project. If you expect more 1:1 time with the advisor, you should choose accordingly...potential hire visits the lab for a full day, gives a 1 hour talk about his or her interests, and spends one-on-one...that supports both paths – you won't know in year 1 what you will want in year 6. Have lab alumni taken... next.” Connie Cepko looks for people who are dedicated and interested in science. “Postdoc hires are ...

...photosensitizer used in this work is composed of two parts: 1) an activatable protein (the FAP - fluorogen activating...reaching greater than 75% photobleaching after just 1 minute of exposure. MG-2I-dL5** experienced minor ...photosensitizers in the comments below!   References 1. He, J., Wang, Y., Missinato, M. A., Onuoha, E., Perkins... encoded photosensitizer. Nature Biotechnology,24(1), 95-99. doi:10.1038/nbt1175. PubMed PMID: 16369538...generation when bound by the dL5** FAP.  The iodine-modifications also shift MG-2I’s excitation wavelength to ...FAP-TAP system works. Find the Plasmids from the Publication Here Light-induced cell ablation with MG-2I-dL5...

...X-linked recessive disorder affecting approximately 1 in 5000 males. DMD is caused primarily by frameshift... applicable to 80% of DMD patients. Figure 1: Exon skipping strategies for Duchenne muscular dystrophy...needed. First, CRISPR delivery must be optimized to: 1. Reach a high percentage of muscle cells throughout...also be closer than we might expect.   References 1. Kaiser J. CRISPR helps heal mice with muscular dystrophy...neurogenetic diseases. Nucleic Acid Ther. 2014 Feb;24(1):69-86. PubMed PMID: 24506781. PubMed Central PMCID...);186-91. doi: 10.1038/nature14299. Epub 2015 Apr 1. PubMed PMID: 25830891. PubMed Central PMCID: PMC4393360...setting. CRISPR is often used for animal germline modification, to repair or add in disease-causing mutations...

... 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...

...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...

...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...

...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 ...

...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 ...

...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 ...

...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...