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...Addgene's Special Collections). Arguably, oncology has benefited most as cancer cells are amenable to culture...misidentified cell lines. The solution Fortunately it has never been easier to conduct high quality cell culture...identity. For example, perhaps a working cell bank has been made by a research institution (an excellent...at the scene of a crime (every human in the world has a unique STR profile) and can also be used as a paternity...topics. The LGC Group also provides standards and has laboratories to carry our independent quality control...

...firm The path to a career as a Grant Professional has been a long and winding path for many in the field...?  In the past 5-10 years, the Grants Profession has become just that, a profession. A grant professional...and the increasing regulatory process for grants has fostered the demand for experts in the field of grants... find your people Find someone who is working or has worked as a Grant Professional to mentor and coach...includes the social media community GrantChat. She has served in the nonprofit and government sector for...

... in situ. Used since the mid-90s, this technique has revolutionised the way we apprehend molecular complexes...protein interactions in living cells. Since then FRET has also been used to measure conformational changes,...brightness - even though in the most popular pair, CFP has five-fold less brightness than YFP. The stoichiometry...Fluorescence lifetime imaging microscopy method FLIM has been developed more recently and is the most rigorous...emergence of faster measurement systems, this technique has still many good days and good “gigs” ahead...No offense...

...policy. Since early March, the open access publisher has required authors to include a note as to where readers...how much data was necessary and reasonable. PLOS has since clarified their data sharing policy and recently...Who is to say that the initial data-generating lab has efficiently trawled their own data? A distinct (perhaps...many have multiple, complex panels. PLOS, itself, has not finalized its definition of necessary data requirements.... Secondly, science is highly technical and this has a natural, albeit unintentional, tendency to work...

... and polling, Blugene's new virus friend finally has a name! We thank you for your participation in suggesting...play on viral vector terms. And, the name Kendall has a special place in our hearts: our first two offices...

...Training as a scientist in the academic system has many pluses. I delighted in my graduate school years...call this building relationships because networking has gotten a bad reputation (as in…”I just hate networking...connections. A mistake made by many novice networkers has a potential to alienate connections. If meeting with...

... Science Guides Addgene has created the following guides to help you start learning...

...targeting. Fast forward a decade, and the CRISPR field has exploded with dozens of Cas enzymes and variants ...scientific community. What are the features? CasPEDIA has collected relevant information on all the Cas enzymes...cleaves both strands of its dsDNA target in cis and has no trans activity. AsCas12a, on the other hand, generates...learn about Cas domains. CasPEDIA either directly has this information or links out to this content and...

...ElizabethKenkel) December 5, 2018 How social media has helped me in science It may have taken a bit of FOMO...Here are three examples of how using social media has helped me or my lab mates: For me, using social ...with social media as a scientist Using social media has enriched my professional development, and while it...helped you fit social media into your busy schedule. Has social media helped you advance your research or ...

...being targeted via RNAi. Recently the Vellai lab has created an improved RNAi vector that leads to significantly...sequence of the toehold switch. For example, Green has also developed toehold switches to detect Zika virus...protein motifs than previously thought. Sidhu’s group has made several hundred SH3 domain containing plasmids.... In addition to these constructs, the Sidhu lab has made hundreds of additional plasmids available through...

...and post-docs alike are wondering where the time has gone. Are we any closer to graduating, publishing...unsure of the next steps. Harvard PI Angela DePace has put personal development at the center of her lab... and postdoc training. On the other side, the PI has an opportunity to check in with lab members, determine...

... field of induced pluripotent stem cells (iPSCs) has been around for 10 years. In that time, scientists... of Myc or other oncogenes after differentiation has been linked to tumor formation in iPSC-derived and...iPSCs compared to other delivery methods. Retrovirus has a cloning capacity of ~8 kb, so either a single polycistronic...factors driven by one promoter. While lentivirus has similar rates of reprogramming when compared to MMLV-derived...mechanism between a vector and chromosomal DNA. PB has a cloning capacity of ~9-14 kb. This system is transfected...cell division. A drawback to this approach is it has lower levels of efficiency at generating iPSCs, usually...reprogram cells is that it’s simple and efficient. It has a high efficiency of reprogramming when compared ...

...wildtype Cas9 with optimized chimeric gRNA has high efficiency but has been shown to have off-target effects... the Genome Engineering Using CRISPR/Cas Systems has expanded out of control. To further consolidate these... a new system, developed by the Zhang lab, which has comparable efficiency to the optimized chimeric design...sequence is present in both alleles. The Zhang lab has found that if a CRISPR is used to engineer a locus...relative to where the homology ends? A9: The Zhang lab has always used homology arms that are less than 15 bp...recombination? A10: The most we've tried to insert so far has been 1 kb. We used homology arms that were 800 bp...for positive ones.  -- OR -- If your HR template has a selection marker such as puromycin, you can (also...

...objective performs essentially the same task but has different magnifying powers. When you look at the...

...to find the perfect combination. The EcoFlex Kit has a wide array of DNA parts that can be assembled into...yeast MoClo is also for eukaryotes! The technology has been used to optimize protein secretion and expression...possible through MoClo. The MoClo Plant Parts Kit has everything you would expect in a MoClo kit plus the... general plant cloning experiments. Addgene also has two additional MoClo plant kits: Plant Parts Kit ...; Chamness et al., 2023). The Plant Parts II kit has a focus on infrastructure with shuttle vectors, several... gene silencing vectors. The Plant Parts III kit has a focus on transformation and genome engineering....Brychcy et al., 2023). For plant biologists, Addgene has not one, not two, but three CRISPR MoClo kits for...

... at the Georgetown University School of Medicine has used this gene editing system frequently over the...in human cells (Kim et al, 2016). The Waldman lab has designed their AAV tagging vectors (pAAV-SEPT-Acceptor...via gene trap enrichment, whereas pAAV-TK-Acceptor has a conventional heterologous promoter-driven TK-neoR...editing compared to CRISPR editing CRISPR, of course, has started a revolution in gene editing. However, while... through aberrant double strand break repair, it has proven more challenging to develop high efficiency...

...reprogrammed less efficiently, no such correlation has been found between the age of the patients from whom...potentially promote tumorigenesis in vivo. The phenomenon has been recorded in past gene therapy trials and remains...reprogramming factors To date, the mRNA transfection method has not been used successfully to derive iPSCs from PBMCs...However, reprogramming of PBMCs by episomal vectors has yielded hiPSCs at a low efficiency (0.001-0.03%) ...efficiency: the pCXLE episomal vector toolkit It has been shown previously that suppression of TP53 enhances...

...quantified by cell transduction assays. Wild-type AAV2 has been reported to have a near-perfect physical-to-...performed on all new viral vector preparations. Addgene has used this method to generally validate our viral ... Material (AAV SFM), which is an AAV sample that has been quantified by 16 labs around the world and can...Hindson et al., 2011, Hindson et al., 2013), which has been shown to be more precise and more repeatable...2011): 8604-8610.4. Hindson CM, Chevillet JR, Briggs HA, Gallichotte EN, Ruf IK, Hindson BJ, Vessella RL,...

...challenge of keeping up on studies in many disciplines has been a long brewing frustration for me. I read a ...citations in their own work. Of course not all research has to be influenced by research in other disciplines...platforms were developing, Google acquired Youtube and has been growing its online video market ever since. ...this point most videos are based on research that has been published, but many discuss in-progress research...videographer, is a retired botanist and oceanographer who has a blog and several videos on sharing your research...

...University of California system, the postdoc union has negotiated a raise from $43,692 to $48,216, an increase...increase in salary of 58.3%. University of Delaware has a minimum of $36,360, requiring an increase of 30.6%...Hospital is the only part of the Harvard group that has responded to the FLSA at the time of writing, and...good thing. Reliance on a cheap trainee workforce has clearly not been a sustainable approach to biomedical...biomedical research in the U.S. and postdoctoral training has not been demonstrated to be required for the jobs...

...into Reddit. Here are some reasons why: 1. Reddit has a large and active community of people interested...right - the Reddit community is active, engaged, and has great questions! 2. The r/Science subreddit is well...the internet, using dialogue). Luckily r/Science has a great team of moderators who facilitated scheduling...preferred time to start answering questions. r/Science has pretty strict rules about not being too sales-y in...the requested topics to the Addgene blog The blog has many other excellent posts on science career topics...

...Samantha Young. The use of CRISPR/Cas9 for gene editing has expanded since its adaptation for use in mammalian...biology surrounding a particular gene (although it has been quite successfully adapted for use in screening... method for validating your selected gRNA’s, and has the bonus benefit of supplying you with primers for...sturdier than the delicate RNA and therefore there has less chance of contamination with RNase leading to...products will give specific details on what exactly has been inserted or deleted. Each lab will use a slightly...

... maintain itself within host cells.  The plasmid has multiple ori that are functionally differentiated...various hosts (Pagatto and Dillon 2001).  The plasmid has its own replication initiation factors and proteins...the host. Host range depends on whether the host has the components to recognize the ori and allow for...restriction enzymes (Meyer et al. 1977). So if a plasmid has few restriction sites, it may be able to sneak past...commonly used bacteria in lab manipulations. Table 1 has a detailed list of the bacterial species these plasmids...

... 16, 2020 by Beth Kenkel.  CRISPR genome editing has quickly become a popular system for in vitro and ...packaging capacity of AAV (~4.7 kb). While this approach has been proven feasible, it leaves little room for additional...the number of targetable loci, and truncated Cas9 has much lower efficiency than its wild-type counterpart...amino acids, can also be packaged into AAV. NmeCas9 has the added advantage that it can be turned off by ...daunting, it’s actually a challenge that the field has overcome with split AAVs. In general split AAVs break...

... and serotype, respectively. For example, AAV2/8 has a type 2 ITR and a type 8 serotype. Almost all vectors... components      Cargo capacity Empty rAAV has a cargo capacity of ~ 4.7 kb, meaning that your gene...usually quite stable and in some mammalian tissues has shown expression for several years (Wonjo et al.,...scAAV vector with half the size of the ssAAV genome has a mutation in the terminal resolution site (TRS) ...

... is an amazing service to biology scientists and has saved countless hours of lab time (in both donating...have at least one plasmid deposited at Addgene that has been distributed more than 100 times.   “I really...ready-to-use viral vector preps. Today, that number has grown to over 114,600 viral preps. And in 2025, we...resources like this are why my brand of neuroscience has exploded. Because we have a resource like Addgene...

...  Disadvantages Every rose has its thorns and every affinity reagent has its downsides. These reduced-in-size...created in lab in situations where a suitable antibody has been identified but a smaller or less immunogenic...using combinatorial libraries, and high specificity has been reported for a number of targets. Due to their...

...developing and optimizing biosensors in the lab. He has recently developed a third-generation intensiometric...release from hundreds of synapses at once. “This work has only been made possible due to the collaborative ... at the German Cancer Research Center (DKFZ) who has developed new redox sensitive GFP2 (roGFP2)-based...Qiu’s work on the PV neurons is in progress, she has validated the dual virus method to study CR neurons...

...nuclease activity, catalytically dead Cas9 (dCas9) has many applications, the most well-known being transcriptional...transcriptional activation/repression. dCas9 has also previously been adapted to fluorescently label a sequence...imaging time for this technique. The NIH Common Fund has made mapping the 4D nucleome a specific priority,...

...Addgene has distributed over 800,000 plasmids since 2004. In that time, our lab has gotten a lot experience...

...promoter of the 5’LTR has been deleted to reduce its activity. A CMV or an EF1α promoter has been inserted in...The risk of generating RCL is low but extra care has to be taken especially if you are working with proto-oncogenes...previously used to drive expression from the LTRs) has been removed, Rev (which facilitates nuclear export...expression. In contrast, in the Tet-on system, tTA has been modified (and renamed rtTA) to bind TRE only...viral binding. This method, called spinoculation, has been found very effective for T cells transduction...

... population level, but each individual cell only has a maximum of one gene knocked out. In this genetically...containing up to around 200,000 different gRNAs. It has been suggested that researchers should use ~400X ...than gRNAs in a screen, to ensure that each gRNA has a good chance to contribute to the screen. In the... If you are lucky and find that a druggable gene has a central role in your system, you could majorly ...guest blogger Fredrik Wermeling! Fredrik Wermeling has a research group at the Centrum for Molecular Medicine...Medicine, Solna, Karolinska Institutet, in Sweden. He has a particular interest in using molecular tools to...

...It is fair to say that CRISPR has revolutionized molecular biology and has enabled functional studies in...enzyme named Dicer (Figure 1). The plant kingdom has expanded its repertoire of Dicer enzymes and these...knockout methods of the same gene. However, recent work has called this prevalent assumption into question. Specifically...multiple target sequences are concatenated. hpRNA has been extensively used in plant research and many ...constructs are available to the community. Each construct has unique features, such as inducible expression of ...

... techniques is a speculation of yesteryear which has been realized recently. Science Fiction (SciFi) writers...even before the discovery of genes themselves.  How has scientific progress in genetic editing impacted the...technology on various non-scientific platforms, CRISPR has also transcended into the world of SciFi. This is...and most important genres of literary fiction. It has always served as a vision of the future and to make...and agriculture, its portrayal in popular fiction has been largely negative. But these negative depictions....1126/science.1258096 Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, Liu DR (2018) Publisher...

...industry just wasn't for me, and as has been highlighted recently it has become more difficult to attain ...interesting and luckily genetics is a sector which has been rapidly growing. These factors left me optimistic...because it is a small and dynamic company and so it has the flexibility of a ‘start-up’. I was surprised ...schedule.  I really love my highly varied role, it has allowed me to travel and to share my passion for ...everyone. My career path may seem quite unusual but it has allowed me to try out working in many different sectors...

...   Fig. 1: SpCas9 has sparse PAM coverage in the human genome. SpRY Cas9 has PAM sites covering the... a PAM with no sequence constraints, or at least has looser or different requirements. Luckily, there ...alternative PAM requirements (Zhao et. al, 2023). xCas9 has been shown to be less effective at NGH PAMs, though...

..., full spectrum flow cytometry (“spectral flow”) has emerged as a particularly useful tool for those wishing...for PE. In order to correct for this, compensation has to be applied to “subtract” this spillover and correctly...they can more easily be detected. Cytek’s website has a convenient tool, called the similarity index, which...beads or cells. Overall, spectral flow cytometry has the ability to generate incredible amounts of data...

...Automating bacterial transformation One protocol that has been automated is high throughput transformation....benefits of the Biomek FxP is it’s customizability. It has a large deck that can house many different types ... how long to keep it there. In addition, the FxP has two robotic, mobile pods - one pod can pick up 96...cap sealer. This is a simple table top device that has a roller to go over caps and push them into micronic...

... development and function. To date, gene therapy has been used to treat 10 infants with this disease. ... nucleic acid cargo (DNA or RNA) to target cells has been challenging. A variety of vector delivery technologies...AAV is not associated with any pathogenicity and has a low immunogenic profile. AAV was first discovered...Harshana S De Silva Feelixge is a researcher whose work has focused on gene therapy for viral infections. She...

..., that informs us that this antibody has specific binding and has a strong signal. We call the unique ...strong signal has a shorter exposure time than the negative control, and a weak signal has a longer exposure...