...repeats (ITRs) are what makes an AAV transfer plasmid an AAV transfer plasmid. ITR sequences are 145 bases...read through this challenging sequence. Transgene The transgene is the gene that will be delivered by the...Most AAV transfer plasmids have a promoter, but this element isn’t required when the transfer plasmid ...control transgene expression. Lox sites are directional 34 bp sequences that can flank the transgene and ...(WHP) Posttranscriptional Regulatory Element (WPRE) forms a tertiary structure when transcribed which ... nuclear export of RNA and RNA translation, and promotes RNA transcript longevity. The polyA is located...proteins needed for the virus to replicate,  and the transfer plasmid which contains the viral genome. In today...

...sybodies against membrane proteins Developing antibodies against proteins embedded in membranes is tricky because...develop antibodies against unstable targets like membrane proteins that may unfold due to camelid high body...synthetic nanobodies, called sybodies, against membrane protein targets in as little as 3 weeks (Zimmermann...lab can quickly select for sybodies targeting a membrane protein of interest using the Sybody Generation...have a concave, loop, or convex scaffold and are randomized with a defined set of different trinucleotides...serines and threonines at positions that can be randomized for creating the libraries (Zimmermann et al.... the process for selection of sybodies against membrane proteins. Image from Zimmermann et al., 2018. ...

...and Harvard Medical School. Scientists making transgenic organisms with Cas9 should be aware of the potential...conducted with extreme caution. Why does making transgenic organisms with Cas9 risk creating a gene drive...drives that can spread through populations of transgenic laboratory organisms but not wild organisms. ...targeting a synthetic sequence will only spread in transgenic laboratory populations with that sequence2. Both...collective technologies are developed safely and transparently.  For more information, visit www.sculptingevolution.org...

...insoluble granules. We overcame this problem by engineering a plasmid which contained cold tolerant chaperones...bacteria out of their optimally suited temperature ranges and make them grow better at suboptimal ones, which...bitter cold.  Since our objective is to induce tolerance towards cold, we decided to focus on a special... of Earth’s harshest environments: corrosive pH ranges, crushing pressures, near-melting temperatures,... host cells (the simplest example would be a transformed mesophilic, for example E. coli, carrying a plasmid...hybrid system where we could incorporate cold-tolerance inducing genes within a regular mesophilic host.... And thus was born ELSA - our happy lil’ cold-tolerant E. coli! Additionally, we’re also working towards...

...third Addgene/Seeding Labs plasmid grant and plans to use the grant to order plasmids she can use as positive...in PCR-based cervical cancer screens. This third grant represents the continuation of our efforts to provide...Bengyella and Kwabena Duedu who were awarded similar grants back in June, Gladys will be using plasmids in ...will be opening the Addgene/Seeding Labs Plasmid Grant to a third round of applicants at the start of 2017...Superhero 5k Find New Cancer Resources Learn About Our Grants to Rare Disease Research Resources on Addgene.... seeding labs plasmid grant...

...The goal of Kiran Musunuru's lab in Harvard's Department of Stem Cell and Regenerative Biology is to ...modeling work in human pluripotent stem cells: a transcription activator-like effector nuclease (TALEN) kit... designer proteins that will introduce a double-strand break in the genome anywhere you would like. Cells...cells after the plasmids have been introduced by transfection or electroporation. All you do to create a protein...genetic backgrounds. It becomes more of an apples-to-oranges comparison. Addgene: Can you compare the TALEN ...the target site. Also, any time you create double-strand breaks, there is a possibility of introducing a...

...type VI-D enzymes are the latest tools in the transcriptome engineering toolbox. How were Cas13d enzymes...small hairpin RNA interference, dCas9-mediated transcriptional inhibition (CRISPRi), and Cas13a/Cas13b RNA...only time will tell how large the genome and transcriptome engineering toolbox will be. It is, however,...community. References Konermann, Silvana, et al. “Transcriptome Engineering with RNA-Targeting Type VI-D CRISPR...Editing with REPAIR Get tips for using Cas9 Transcriptional Activators Learn about Cas13a (C2c2) Characterization...

...19329793. PubMed Central PMCID: PMC2719376. 5. Barany, Francis. "Genetic disease detection and DNA amplification...1986365. PubMed Central PMCID: PMC50775. 6. Barany, Francis, and David H. Gelfand. "Cloning, overexpression...Central PMCID: PMC208194. 8. Luo, Jianying, and Francis Barany. "Identification of essential residues in Thermus...(1989): 245-254. PubMed PMID: 2753355. 12. Barany, Francis. "The ligase chain reaction in a PCR world....25. Luo, Jianying, Donald E. Bergstrom, and Francis Barany. "Improving the fidelity of Thermus thermophilus...temperatures (up to 95 °C) and is active over a range of elevated temperatures (37–75 °C). T4 DNA ligase...temperature near the melting temperature (Tm) of the DNA strands. This selectively reduces the concentration of ...

...fused to a fluorescent protein. “Frankenbody,” like the infamous frankenstein, was constructed by grafting...called this probe the HA frankenbody (Zhao et al., 2019).  To develop HA frankenbody, the Stasevich lab tested...can you do with the HA frankenbody? Another large advantage of the HA frankenbody is that it remains bound...also proved that HA frankenbody can be effectively used to track single mRNA translation in U2OS cells. HA...acids, thus it is translated quickly by the ribosome. This feature provides HA frankenbody the ability to...properly once inside cells.  Developing the HA frankenbody To combat the issues with these antibody based...lab observed colocalization of GFP tagged HA frankenbody with an mCherry, 4xHA tagged histone protein ...

... recent works, transdifferentiation is usually carried out by expressing transcription factors specific...successfully transdifferentiated into neurons and β-cells [40-42]. Reprogramming and transdifferentiation: When...redifferentiated to a different cell type. Transdifferentiation In this process, differentiated adult somatic...plasmid electroporation (episomal) [7, 8] and mRNA transfection [9]. Many of the plasmids used for these methods...variety of plasmids for differentiation here. Transdifferentiation Dedifferentiation to an intermediate pluripotent...primitive pluripotent stem cell state, through transdifferentiation adult somatic cells are converted directly...newt over 100 years ago [36]. While natural transdifferentiation is rare in mammals, an example is observed...

...phosphatase-accessible. Kinase Translocation Reporters (KTRs) Kinase Translocation Reporters (KTRs) are genetically...particularly important for regulating signal transduction and measuring kinase activity at the single-...provides an alternative tool with their Kinase Translocation Reporters (KTRs) whose cellular localization...activity is enhanced, leading to cytoplasmic translocation of the reporter. As a result, the KTR is shuttled...nuclear export and import rates can influence KTR translocation. Work from the Covert lab suggests that these... negligible effect on KTRs, however nuclear translocation rates are another variable to consider when ...measurements of kinase activity in single cells using translocation reporters. Nature protocols, 13 1, 155-169. ...

... successfully mediated transduction and expression of the full-length transgene in vitro and in vivo (...this, larger functional transgene products were produced following transduction.  So what’s going on here...between part A and B of the transgene to reconstitute the full-length transgene (Chamberlain et al., 2016...cells in mice. This vector transduced ~10% of cells, similar to the transduction rate of a single AAV. Odom...muscle physiological performance.  2. Trans-splicing Overview: For trans-plicing, splice site donor and acceptor...reconstitute the two pieces of the transgene.The AAV encoding part A of the transgene contains a promoter and the...fragment of the transgene, followed by a splice site donor. The AAV encoding part B of the transgene contains...

...expressing a novel reverse transcriptase (RT), MarathonRT. Reverse transcriptases are critical tools for ... to transform and express in bacteria, which, in turn, makes them more adaptable to a broad range of applications...!  MarathonRT: A highly processive reverse transcriptase Article contributed by Will Arnold  Listen ...RTs in this regard and is even able to reverse transcribe the extremely long, highly structured HCV genome...structured template!). MarathonRT also faithfully transcribes RNA to DNA with low error rates compared to other... are dependent on long, error-free, reverse transcription of RNA such as long-read cDNA sequencing and...29109157. Nanobody toolkit to monitor retrograde transport at the cell surface Article contributed by Marcy...

...Addgene-Seeding Labs Plasmid Grant Check Out Previous Addgene-Seeding Labs Plasmid Grant Awardees Listen to Our...Labs and Addgene. The Addgene-Seeding Labs plasmid grant is a collaborative endeavor providing plasmids to...who were awarded the Addgene-Seeding Labs Plasmid grant this year. These scientists work on projects spanning...and to increase the potential for this drought tolerant plant, Dr. Robertson aims to make use of the CRISPR... different CRISPR applications in plants.     Transgenic yeast as source of dietary supplements Dr. Mrinalini... proteins. In this project she aims to create transgenic yeast as an alternative source of essential omega... diverse groups of scientists working in a wide range of research fields and geographical locations. We...

...plasmid page here). Now I spend much less time wrangling bacteria and instead help Addgene fulfill its ...better and make scientists’ lives easier. Why transition to Addgene? So why did I join Addgene? Was I ...easier. I’m hoping my story demonstrates that transitioning from academia doesn’t have to mean abandoning...management, information distillation, etc) are transferable to many different fields. If you start thinking...

...received a grant from the National Institutes of Health that will address this problem.The grant, which is...within the next year. NIH grants and Addgene’s mission This is the first NIH grant that Addgene has received... of California Davis School of Medicine on this grant. Dr. Trimmer has created thousands of high-quality...NIH’s vision and support for this project. This grant will allow Addgene to further our mission of scientific...working with the community to create a fully transparent resource where researchers can easily share, ...project will be supported by a NIH BRAIN Initiative grant (NS119916). The content of this article is solely...P, Frenzel A, Wong J, Koh XY, Eng H-Y, Lane D, Lefranc M-P, Clark M, Dübel S (2018) When monoclonal antibodies...

...engineering using the CRISPR-Cas9 system. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang...another transfection method such as cationic liposome-based transfection. Optimize transfection conditions... (Crick) strand while sgRNA-B’s protospacer sequence and PAM fall on the top (Watson) strand ( Figure ..., which is used for MEL cells. However, transient transfection of CRISPR/Cas9 plasmids may be successfully...single guide RNAs (sgRNAs) to create two double strand breaks (DSBs) at a locus in order to delete the...leading to nonsense mediated decay of the mRNA transcript ( Figure 1B ). Use the example guides for the...protospacer sequence/PAM relative to the top or bottom strand. Determine the reverse complement of each guide...

...fluorescent protein transcription in response to river contaminants and transform these plasmids into ...Addgene-Seeding Labs Plasmid Grant Check Out Addgene-Seeding Labs Plasmid Grant Awardees in 2016 and 2017... specifically, the Addgene-Seeding Labs plasmid grant provide plasmids to these researchers. In the third...third round of the Addgene-Seeding Labs plasmid grant we are excited to introduce the 5 awardees and their...into a bacterial strain. The sensor will be transported inside a small 3D printed robot that will “swim” ... diverse groups of scientists working in a wide range of research fields and geographical locations. In...see how all of the Addgene-Seeding Labs Plasmid Grant awardees' projects progress. Subscribe to the Addgene...

...course much more difficult than in cell culture, but Ran et al. have shown that CRISPR and AAV can be used...Thakore PI, Moreb EA, Rivera RM, Madhavan S, Pan X, Ran FA, Yan WX, Asokan A, Zhang F, Duan D, Gersbach CA...WL, Widrick JJ, Yan WX, Maesner C, Wu EY, Xiao R, Ran FA, Cong L, Zhang F, Vandenberghe LH, Church GM, ...: 25692716. PubMed Central PMCID: PMC4335351. 9. Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz...) to remove mutated exons from the dystrophin transcript. Unfortunately, the oligonucleotides only modestly...in one experiment, they found the exon-skipped transcript constituted 59% of total dystrophin mRNA, similar...low to no off-target activity at the ten highest-ranking predicted off-target sites. Each paper characterizes...

...874 . https://doi.org/10.1038/nmeth.3993  Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu.../doi.org/10.1038/s41586-019-0908-x  Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, Ishitani...949. https://doi.org/10.1016/j.cell.2014.02.001  Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz...gene delivery due to their low immunogenicity and range of serotypes allowing preferential infection of ...prime editors. While using AAV to deliver large transgenes might seem daunting, it’s actually a challenge...split AAVs. In general split AAVs break a large transgene in two pieces and package each piece into an individual... individual AAV. When a cell is transduced by both AAVs, the full length gene and/or protein is reconstituted...