...applications. Figure 1: Schematic of an RNAi-inducing transgene. Typically, the desired target of RNAi is...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...modifications of complementary gene sequences (Figure 1). As a result, gene silencing can be easily induced...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...eukaryotic process where approximately 20-30 nucleotides of double-stranded RNA (dsRNA) results in downregulation...

...Transposons: The jumping genes. Nature Education 1(1):204. https://www.nature.com/scitable/topicpage/transposons-the-jumping-genes...poly(A). ORF2 encodes endonuclease and reverse transcriptase activities.    Figure 1: Overview of...cholerae that encodes a Type I-F CRISPR-Cas system. This system has four major components: 1) a CRISPR RNA... 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... sequence that moves around. This DNA sequence encodes the proteins the transposon needs for transposition...contain two genes: gag and pol. The pol polyprotein encodes the reverse transcriptase and integrase enzymes...

...experiments. A recent publication [1] from the Sánchez lab describes the plasmids and protocols required...available through the deposit) are described in the Sánchez lab publication [1] so that the students performing...can easily investigate in planarians. References 1. Alice Accorsi, Monique M. Williams, Eric J. Ross,...Proceedings of the National Academy of Sciences 100.suppl 1 (2003): 11861-11865. PubMed PMID: 12917490. PubMed...companion resource called “cutting class” which provides more detailed materials and protocol resources...synthetically made RNA (riboprobes) with modified nucleotides (for example, Digoxigenin-labelled Uracil) that... molecular cloning to insert the cDNA into the desired plasmid backbone. The Sánchez lab plasmids allow...

...discovered such as Kaede and PS-CFP2 (see Table 1). Table 1: Properties of Selected Irreversible Photoactivatable...preserve cellular structures (0.5-1% osmium tetroxide). These conditions destroy most PA-FPs, but, in 2015, ...) 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...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 ...to green). For example, mEosFP (named after the goddess of dawn in Greek mythology) switches from green...

...2017). Within an interior cavity, Cas9 grips nucleotides 1-10 of the guide RNA, prearranged for base pairing... basic parts of the Cas9 enzyme.   Figure 1: A cartoon depiction of Cas9’s two major lobes, REC...the CRISPR system (Nishimasu et al., 2014). Table 1. Summary of Cas9 domains, the lobe each domain belongs...state (PDB ID 4CMP) with domains colored as in Figure 1. HNH, red; RuvC, orange; CTD, yellow; REC lobe, light... on itself at the bottom of the image. Stem loops 1 and 2 extend horizontally across both lobes. The guide...outside of the protein, spanning both lobes. Stem loop 1, nestled between the REC and CTD domains, is required...343–347. https://doi.org/10.1038/s41586-022-04470-1 Cofsky, J. C., Soczek, K. M., Knott, G. J., Nogales...

...acceptor fluorophore. The typical steps in FRET are: 1. Donor fluorophore excitation by absorption of a photon... overlap, the fluorophores must be located within 1-10nm of each other and be oriented appropriately for... from various papers to make the decision easier. 1. Müller, Sara M., et al. "Quantification of Förster...diseases and even assist in drug discovery. References 1. Baird, Geoffrey S., et al. "Circular permutation ...Resonance Energy Transfer (FRET) was originally described by Theodor Förster in 1948 as a variation of the... observed light emission by fluorescence. The widespread use of FRET with fluorescent molecules, including...FRET probe, try searching PubMed for articles describing the FRET tool that you are looking for and check...

...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...(s) Cell lines Cell lines, primary cells Table 1: Key differences between original CAPTURE and CAPTURE...The Xu lab presented three ways to use CAPTURE 2.0: 1. Multiplex capture of chromatin interactions The Xu...Variant for Your Next Experiment Learn More About Designing gRNAs Against Your Favorite CREs Here Additional...

...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...Maria were encouraged by previous reports that described magnetic responses of cryptochrome proteins or...

...118). Humana Press. https://doi.org/10.1385/1-59259-301-1:99 Resources on Addgene.org Addgene antibodies...antibody detection is desired… and yes, we have an antibody for that!   Fig. 1 - Diagram of His-tagged...You’ve designed the perfect experiment – controls, conditions, and everything in between – now all you...This is where affinity tags come in – they are peptides which do not naturally exist in most expression...protein for downstream applications Below, we describe a few of the most common tags available. Maltose...antibody is an alternative to amylose resin and also provides a visual detection mechanism via applications ...

...collected from heterozygous individuals. Only 1/4 of the descendants of the heterozygous plants, therefore, ...in all other plants, anthocyanins.   Figure 1: An adult plant expressing RUBY. Photos by Andrew ... (2020). https://doi.org/10.1038/s41438-020-00390-1 Additional resources on the Addgene blog  CRISPR 101... Antibiotic selection would only then kill the desired plants! PCR or fluorescent markers are currently... have similar drawbacks, often requiring the destruction of plants or tissue for GUS staining or are not...from these plants will be green, containing the desired mutation but lacking both RUBY and the construct...compared to other markers lends itself well to nondestructive selection. If RUBY was integrated into a CRISPR...

... emailed submission) in the Deposit menu: Figure 1: Begin the deposit process by clicking "Submit Online...cases Please contact us at [email protected] or +1 (617)-ADDGENE (233-4363) if your deposit falls into...Addgene?" or contact us at [email protected] or +1 (617)-ADDGENE (233-4363) with additional questions...strain. Please contact us at [email protected] or +1 (617)-ADDGENE (233-4363) if you’d like to distribute...page so that any data you enter is not lost.   Step 1: Sequences, Maps, and Files Please provide any full...a 1.5 mL microfuge tube at a concentration of 0.1–1 µg/µL (or bacterial streaks, if you prefer). If you...preprint title or a general description of the deposit. Select your desired distribution status: Hold...

...occur. While the Brainbow-1 system employs Cre-mediated deletion, the design of Brainbow-2.1 cleverly ...homologous, preventing design of antibodies specific to each fluorophore. Third, Brainbow-1 and Brainbow-2 each...the Brainbow mouse neural labeling system. Brainbow-1 and -2 have been further refined to create Brainbow...dendritic processes not previously visible with Brainbow-1 and -2. The general structure of Brainbow-1.0 is retained...Rheenen J, Simons BD, Clevers H. Cell. 2010 Oct 1;143(1):134-44. PubMed. Drosophila Brainbow: a recombinase-based...R26R-Confetti described in Hugo J. Snippert, et al. (2010) and the MAGIC Marker strategy described in Karine...immediately following the promoter. The STOP cassette includes a mutant YFP that does not fluoresce, but can ...

...detector for that fluorophore. For example, in figure 1, the FITC signal is spilling over into the primary...spectra to use for compensation.     Figure 1: The emission spectra and filters for FITC and PE ...in the panel. This complexity index ranges from 0-1, with a value of “0” indicating those markers are ...completely distinct from one another, and a value of “1” indicating those markers are identical and completely...positive and negative populations, Careful panel design In both conventional and spectral flow cytometry...indistinguishable. This tool is of particular value when designing a panel.     Figure 3: Similarity index...if markers are co-expressed in your cells when designing your panel. If you have two highly overlapping...

...Sullender M, Ebert BL, Xavier RJ, Root DE (2014) Rational design of highly active sgRNAs for CRISPR-Cas9...Last updated on Oct 1, 2020 by Aliyah Weinstein. This post was contributed by guest bloggers, Wenning...process check out our Mouse Modeling blog posts: Part 1: Genetically Engineered Mice and Part 2: Breeding ...LO, Morgan JL, Juan WC, Roca X, Ong ST, Bergstrom DE (2018) Viable Mice with Extensive Gene Humanization... mouse genome is made up of 3 x 109 nucleotides (nt), and encodes 23,000 or so genes. It would be great...guide you in gRNA design. We have been using Benchling, but there are many other gRNA design tools available...for cells that had successfully incorporated the desired mutation, and then derive mice from these engineered...

...attract fish for them to prey upon (Figure 1). Figure 1: The sea anemone's red color can be used ...synthetic biology. Journal of Biological Engineering, 12(1). https://doi.org/10.1186/s13036-018-0100-0 Murakoshi...expression in living cells. Scientific Reports, 9(1). https://doi.org/10.1038/s41598-019-48604-4 Salih...fluorescent proteins, it’s important to note that despite their distinction by name, chromoproteins are actually...chromoproteins have a shorter history. In 1987, scientists described an unusual blue-colored protein from the jellyfish... chromoprotein plasmid that combines the most desirable features: intense color, fast maturation, and ...recombination events: The plasmid carrying the desired genetic modification integrates into the genome...

.... Table 1 provides an overview of pooled library protocols you can use as a reference. Table 1: Examples... be more difficult to transform.    Figure 1: Factors to consider when creating a pooled library...etc. The DNA amount can be anywhere from 10 ng to 1 μg of DNA. It often correlates with the size of the...(Are the inserts of different sizes?), and the desired coverage for the library (How important is it that...  The most important factor to consider is your desired library coverage, since this will help decide what...should always experimentally test the protocol you design to ensure that it works for your library before...Addgene. If you have any specific questions about designing your library amplification protocol, feel free...

...reporter gene, which replaces the targeted gene and provides a way to track successfully engineered cells. ...genotype each animal to identify the ones with the desired genotype. Inducible and conditional systems Conditional...

...on-target and off-target editing efficiency Figure 1: Effects of (A) double and (B) single base pair gRNA-target...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...fits into the genome editing landscape. References 1. Kim D, Kim J, Hur JK, Been KW, Yoon SH, Kim JS. Genome-wide...for Cas9 (42 nt vs 100 nt.) However, the newly described non-CRISPR protein NgAgo is smaller than any of... of these options (2.7 kb with 24 nt guides), so it has the potential to eclipse any of the CRISPR nucleases...to examine Cpf1 off-target editing. First, they designed gRNAs with single and double mismatches throughout...

...against the limitations of the assay.      Fig. 1: A schematic representation of Sybr Green chemistry...Methods Mol Biol. 2018;1768:143-160. doi: 10.1007/978-1-4939-7778-9_9. PMID: 29717442. Arya M, Shergill IS...amplification of Plasmodium falciparum multidrug resistance-1 gene (pfmdr1). J Parasitol. 2011 Oct;97(5):939-42....gamma receptor genes FCGR3A and FCGR3B. PloS one, 10(1), e0116791. https://doi.org/10.1371/journal.pone.0116791...results can be affected by DNA quality and primer design, particularly for amplicons under 100 bp (Ma, 2015... concern, look for the lowest-cost option that provides you confidence in the results you need.  Whichever...by running the product on a gel. And only ddPCR provides absolute quantitative data, instead of relative...

... vectors are: 1) the potential generation of replication competent virus [usually HIV-1]; and 2) the potential...systems have been developed over the years based on HIV-1; however, the 1st-generation vectors are never used...which has been shown to repress expression of HIV-1 LTRs (more information is available here). 2nd vs...those seen when using a plasmid backbone that was designed specifically for transient expression (find some...expression of viral constructs is a useful tool as it provides a means to quickly check that a construct is functional...3rd-generation transfer plasmids have a chimeric 5'LTR that includes a CMV or RSV promoter as well as a portion of ...those three genes plus tat. The envelope plasmid provides a heterologous envelope for pseudotyping and is...