...bottleneck is that most recurrently mutated cancer genes are rare and most of the individual variants found...as evolutionary conservation, or predicted deleteriousness of the amino acid change based on maps of protein...scale, broadly available collection of mutant cDNA clones corresponding to cancer variants found in genome...genome sequencing studies (as well as reference cDNA clones mapping to well-annotated cellular pathways). It...These include 634 sequence confirmed mutant cDNA clones corresponding to cancer variants derived from analysis...collaborators to aggregate a sequence-confirmed series of clones corresponding to known cellular signaling pathways...Pathway Reference Set. These Gateway-compatible entry clones are the product of many years of work and we’re...

...technology has changed how scientists edit and control genes, but according to the Broad Institute's Silvana ...technical leap from mutating and deactivating a gene or genes to selectively activating them with the CRISPR system...Konermann said. “If you want to activate all 20,000 genes, it is not possible to test each one and make sure...you are really confident about working for most genes and individual targeting modules. Those two things...for the transcriptional activation of endogenous genes and it consists of three components: 1. A nucleolytically...colleagues demonstrated the new system by screening for genes that, when turned on, make melanoma cells resistant...for each transcription start site in 15,977 human genes. The transcriptional repressor (CRISPRi) library...

...combination with different genes. Having CRISPR systems for assessing genes in defined contexts would ...Annotation of genes in immune cells typically involves the creation of germline knockout mice, which ...the constitutive, ubiquitous knockout of single genes in immune cells in vivo (LaFleur et al., 2019a and...knockouts in their immune systems. However, many genes have different functions in different cell types...Mouse Strain C-CHIME Knocks out a pair of genes pXPR_219 Violet-excited GFP (vex) Rosa26-Cas9...; Rosa26-Cas9 S-CHIME Knocks out pairs of genes sequentially pXPR_220 Violet-excited GFP (vex...These systems were benchmarked on positive control genes in CD4+ and CD8+ T cells. To demonstrate the power...

...choose from when looking to activate genes across diverse cell lines.   dCas9-VP64 Description CRISPR activation...loci across Drosophila cell lines (Sajwan et al., 2019). SPH SPH combines components of SAM and SunTag...variant lacking its endonuclease ability, to bind to genes without editing the genome (Qi et al., 2013). To...exhibits modest levels of gene activation, with some genes experiencing around 2-fold activation levels. Although...HS1 and p65). Performance When targeting single genes, SAM consistently shows the highest levels of gene... multiplex gene regulation (activating multiple genes at once), however, SAM exhibits activation levels...design (SAM) or peptide design (SunTag). This streamlines its delivery, making it a common choice for CRISPR...

... own money at this business and started things up for real: bank account, business cards, incorporation...issues about continuing with the business. We ended up applying to a business accelerator freely stating that...how to run a business. SynBio Axlr8r thought color-changing flowers was a great business idea as well,...social media comments were more often along the lines of “I hate GMOs, but what you are doing makes sense...GMOs that result in a new utility, was there a business opportunity out there for us? After about a month...research and another $30,000 in mentorship and business training. The money is a drop in the bucket against...to develop the technology we will need, and the business training to make the most of it. It has also allowed...

...transgene but also that of adjacent cellular genes. Q7: Which genes are deleted or modified to result in replication...1]; and 2) the potential for oncogenesis through insertional mutagenesis. These risks are largely based...lentiviruses and gamma-retroviruses use the same genes for packaging (that's gag, pol, and env), the isoforms...specifically for transient expression (find some useful backbones here) or from viral transduction. While not ideal...cell line is used; several common laboratory cell lines including 293 were immortalized with adenoviral ...3rd-generation transfer vectors apart? A3: Addgene defines 2nd- or 3rd-generation transfer vectors based on...expression of lentiviral vectors in producer cell lines. Identifying less toxic envelope proteins or envelope...

... identified ricin toxicity genes. The screen also discovered several genes previously not associated with...-throughput systematic approach for identifying genes involved in a biological process. These screens ...per gene and targets all ~20,500 protein-coding genes. Unique, non-overlapping sites in the genome were... pooled library via a lentiviral vector to cell lines that either stably expressed Cas9 or that had been...toxicity screen since essential and non-essential genes for both pathways had previously been identified...identifying >88% of previously identified essential genes while previous Cas9 and shRNA library screens only...only identified  60% of the essential genes. The ricin toxicity screen had a 10% false discovery rate ...

...multiple genes with Gal4/UAS system One can also use Gal4 to drive expression of multiple genes under the... Drosophila geneticist’s main workhorse to turn genes on or off. Gal4 is a transcriptional activator that...machinery to the site to induce gene expression. Thus, genes and siRNA encoded downstream of the UAS sequence...promoter, and Bloomington’s Stock Center has fly lines expressing Gal4 under a variety of promoters resulting...quickly mate your UAS-YFG line with a variety of Gal4 lines and study gene expression in many different tissue...sequences. For example, if you discovered five new genes and wanted to express them within a specific neuronal...dosage effects. For example, overexpression of two genes by one Gal4 driver will produce roughly half as ...

...disadvantages of luminescence versus fluorescence.  Table 1: Properties of Luminescence versus Fluorescence... the emission of photons. The most familiar bioluminescent organism for most people is the firefly (Photinus...surprisingly it is also the most commonly used bioluminescent reporter. This beetle emits a yellow-green ...as substrates. Production of photons by this bioluminescent reporter occurs slower than fluorescent-based...the different mechanisms to produce photons, chemiluminescent reporters are generally less bright than fluorescent...Fluorescence   Luminescence Fluorescence Source of Emitted Photons Chemical reaction High-energy photons...Photinus pyralis) is the most frequently used bioluminescent reporter, other luciferases have been identified...

... They share about 70% of their genes with humans and 85% of human genes associated with a disease have...Zuber, Johannes, et al. "Mouse models of human AML accurately predict chemotherapy response." Genes & development...researchers around the globe. These organisms share many genes with humans, are easily maintained in the lab, and...manipulated with tools like CRISPR to make transgenic lines. The fruit fly (Drosophila melanogaster) Another... of Drosophila research. Morgan discovered that genes were found within chromosomes using the fruit fly... as it is estimated that 75% of disease causing genes in humans have a functional homolog in the fly (...packaged into chromosomes and about 23% of yeast genes have a counterpart in humans (Liu et al., 2017) ...

...replaced S. pyogenes Cas9 in the ABE complex with PAM-variant Cas9 proteins: the S. pyogenes NG-Cas9 (PAM...While editing single genes is very useful, sometimes, you need to modify multiple genes to generate a cellular...adenine base editors with improved editing in cell lines Nicole Gaudelli, the Associate Director and Head...ABE8 constructs using a catalytically “dead” S. pyogenes Cas9 (dC9-ABE8.x-m/d). The dC9-ABE8.x-m/d constructs...adenine base editors The most commonly used S. pyogenes Cas9 requires the target site in the genomic DNA...(“N” can be any nucleotide base, followed by 2 guanines). This limits the use of ABEs as DNA sites that...In addition to stem cells the authors targeted 6 genes in primary human T cells using 8 selected ABE8s ...

...carrying genes for bacterial toxins like colicin typically also carry the necessary resistance genes to help...cell to another). Bacteria need special fertility genes to be capable of conjugation, and these are encoded...organisms to thrive in competitive environments. The genes encoding these functions form the basis of many ... Resistance (R) plasmids: microbial defense Genes allowing bacteria to survive toxins are the clearest...Nevertheless, the transfer of plasmids carrying these genes between bacteria can also have serious implications...: microbial infection Virulence plasmids encode genes that allow the host to infect other organisms. If...difference is virulence plasmids! This category includes genes that help bacteria invade hosts, including attaching...

...from Streptococcus pyogenes.   Figure 2:  Crystal structure of S. pyogenes Cas9 in the apo state... we can see why: two arginines make base-specific hydrogen bonds to the guanines, but the N (a thymine...    Figure 3: Crystal structure of S. pyogenes Cas9 bound to guide RNA (PDB ID 4ZT0), shown opaque...the protein, spanning both lobes. Stem loop 1, nestled between the REC and CTD domains, is required for...protein portion of the CTD, ready to check for PAM sequencines. This helps explain why Cas9 can’t locate PAM...sequence isn’t in place yet.     Figure 4: S. pyogenes Cas9 in the apo state (PDB ID 4CMP, top) compared... residues in the CTD can get cozy with DNA. S. pyogenes Cas9 recognizes the PAM sequence 5’-NGG-3’, where...

...with other genes. He will use fluorescent proteins to monitor the localization of these genes, CRISPR to...to identify candidate genes as drivers of peritoneal mesothelioma tumorigenesis. They plan to use CRISPR...derived peritoneal mesothelioma cells lines and assess their tumorigenesis. These experiments will serve to...validate these candidate genes and allow Nabavi and colleagues to prioritize which genes they will use to develop... Di Schiavi will be able to better choose which genes and pathways to target in future small molecule ...molecules targeting SMN1 and its partners in mouse cell lines. Dr. Di Schiavi hopes that these studies will accelerate... research funding opportunities. References 1. Jones, C., et al. "PP09. 1–2352: Systematic review of ...

...This post was updated on Aug 20, 2020. What genes are important in your phenotype of interest? Many scientists... cause is not entirely known. Identifying which genes are important for a phenotype can lead to a wealth...experiments investigating the role of individual genes or entire pathways in a particular disease process... can also be used to activate or repress target genes without permanently modifying the genome. If you'd... is to use Cas9 and a pool of gRNAs to identify genes that are essential for a given phenotype. Image ...screen a population of mutant cells to identify genes involved in a particular phenotype. CRISPR can be...created to knock out, activate or repress target genes by combining a gRNA library with the aforementioned...

...interspaced palindromic repeats) Cas (CRISPR associated genes) system. This popular genome engineering tool offers... be incorporated into the bacterial genome. Cas genes, which code for polymerases, nucleases, and helicases...adaptive immune system that utilized various cas genes not only to store a record of invading phages but...CRISPR/Cas is the system that scientists have harnessed for genome engineering. The diversity of the CRISPR... CRISPR/Cas systems provides powerful lines of defense against invading phages. Rising to the challenge...challenge - Evolution of anti-CRISPR genes in phages The CRISPR/Cas adaptive immune system seems like the winning...nucleases from re-identifying it (3). Surprisingly, few genes have been identified that neutralize CRISPR/Cas....

...of a gold standard set of genes (1,580 essential and 927 non-essential genes). The Root and Doench lab...viability. In addition, high copy number genes may mimic viability genes as they are subject to an increase ...help reveal the function of lowly expressed genes or genes that are more drastically affected by gene ...overexpression library identified several overlapping genes, most genes were unique to the individual libraries. These...be used to knock out, inhibit or activate target genes by combining specific sgRNAs with Cas9 or Cas9 derivatives... CRISPR libraries in the depletion of essential genes. In the subsampling analysis of the sgRNAs, Brunello...discriminate between non-essential and essential genes similarly to the Brunello knock-out library while...

...with the E1 and E3 (Early 1 and Early 3) genes deleted. These genes’ functions are rescued by helper plasmids...will be expressed, since the early genes are absent, and the late genes (and their structural proteins, ...the world, hypotheses began to develop that this illness was caused by either SARS-CoV-2, Adenovirus type... rarely cause severe disease in humans (and no vaccines are available on the commercial market). Although.... Its genome consists of “early” (E) and “late” genes, and the transition between early and late infection...replication (Harrach, 2014). Given that the early genes encode proteins essential to initiating the whole...plasmids used during production and/or by genes present in the HEK293T cell line used (which have fragments...

...that this system uses plasmids where the genes (either viral genes or minigenome) are under control of a ...transcription (6). The EBOV genome contains seven viral genes, each flanked by a transcription start signal sequence...replication and transcription (Figure 1). All viral genes are removed and replaced by a single (non-viral)...replicates the viral genome and transcribes viral genes. Four viral proteins are required for viral genome...When used to study minigenomes, these four viral genes are each encoded on separate plasmids and are co-transfected...alongside a plasmid encoding the minigenome. These genes and the minigenome are transcribed (and the viral...is technically possible to encode all four viral genes in the same plasmid (as opposed to four separate...

...harmful genes into a transfer vector. Lentiviral particles expressing proto-oncogenes or oncogenes for instance...DNA efficiently into dividing immortalized cells lines but reach their limits when it comes to transferring...packaging plasmid containing all the structural genes necessary for the production of a viral particle...the packaging plasmids lacked the psi signal, the genes encoding the structural proteins could not be integrated...plasmid), 2) the HIV-1 genome containing all viral genes with the exception of the env gene (packaging plasmid...taken especially if you are working with proto-oncogenes or with human samples that have not been tested...generation because this vector requires only three HIV-1 genes (gag, pol, and rev) for production. Third generation...

...studying cell division. However, after 6 years in the business of western blots and cloning, I decided to pack...with little emphasis placed on the marketing or business background of the applicant. However, although...push not only my academic background but also my business acumen and sales experience. Most scientists think... I tore up my “Scientific CV” and wrote a more business friendly CV that would focus on my achievements...conferences I presented at and placed them under headlines entitled “Presentation Experience”, “Career Achievements... profile, matched it with my CV and added more business-like elements. At this point you may be asking... the focus of your CV from  science-focused to business-focused?" The reason is that I knew that the mind-set...

...CRISPR/Cas9 for gene disruption in M. abscessus  Bioluminescent reporter of mechanical forces   Epitope tag...expression levels comparable to GFP during early embryogenesis (Figure 3). mScarlet-I3 also offered clearer...development. Representative images of single focal planes through the middle of each embryo are shown, comparing...elegans: mScarlet3 for applications needing intense brightness (such as low-expression proteins of interest)...Here, the researchers were able to target multiple genes in M. abscessus either simultaneously or stepwise...e0033523. https://doi.org/10.1128/jb.00335-23.  Bioluminescent reporting of mechanical forces with PILATeS...expressed separately) assemble and catalyze the luminescence reaction (Figure 5). The researchers used the...

...plasmids containing these genes. As with the PIs above, many of these genes were discussed at the 2018...context of chromatin?” Though it’s true that adding histones and chromatin architecture to every diagram portraying...off point. The following lists of PIs, articles, genes, and blog posts are not comprehensive, so just because...Zhang Leonard Zon  You!     Chromatin-related genes with plasmids available from Addgene The links below...Keystone meeting for Chromatin. Note that histone genes frequently have many different variant names, not...our website. In-exhaustive Chromatin-related genes with plasmids on Addgene.org AGO1 BRCA1 CREBBP... RBX1 STAG2 Xist Your favorite gene! SWI/SNF Genes:       BRG1(SMARCA4) BAF170 (SMARCC2) ARID1A...

...tested in different plasmid backbones that varied in size or carried different genes. In these cases, gene ...three genes whose proteins are needed sequentially. When Segall-Shapiro et al. placed the genes under ...Researchers express genes of interest from plasmids in order to study gene function or to engineer cells... fluctuations due to copy number by introducing genes of interest into the chromosome. But this has its...replication more than once per cell division and thus, genes closer to the origin of replication will be overrepresented...initiated before the cell has divided. This means that genes located near the origin will be over-represented...stabilized promoters were equivalent whether the genes were expressed from the genome or a plasmid. Thus...

...nitrocellulose membranes and exposed to IPTG in order to induce protein expression. Cells on the membranes will ...high salt.  To probe membranes with target bacterial cells, wash your membranes with PBST to remove salt...done, this technique can also help pinpoint the genes within a sequenced genome that encode for functional... vector. In order to reduce the chances of some clones being outcompeted by others in liquid culture, ...When your protein induction is done, peel the membranes off the plates and place each onto a lysis solution-soaked... gene encodes that protein! First, block your membranes with BSA or skim milk as you would for a Western...to remove cell debris. Then, UV-irradiate the membranes for 15 min to kill any remaining live E. coli....

...cell lines? One of the major uses of the piggyBac™ system is to create stable transgenic cell lines. The...method of making stable cell lines is to transfect, select, and isolate clones. This procedure is cumbersome...expression of certain genes, such as those that have negative impacts on fitness. One consideration when...including making transgenic cell lines and genetically-modified cell lines. Furthermore, the use of piggyBac...be used for numerous applications, including transgenesis, gene-trap screens, and gene editing. How does...genetic manipulations in animal models or cells lines, including stem cells, without leaving behind any... somewhere between one hundred and one thousand clones to find a cell line with the edit of interest. ...

...different Dicer-like enzymes are involved in the biogenesis of different small RNAs (Figure 1). These siRNAs...elicits immunogenic reactions that complicate the effectiveness and interpretation of RNAi. Fortunately, researchers...incorrectly relegate RNAi to history. The straightforwardness of RNAi is unmatched—unlike CRISPR experiments...the level of DNA, thereby limiting CRISPR’s effectiveness at high copy numbers and genomic loci with low...strategies cannot be used to investigate essential genes. Historically, gene silencing methods were thought...equivalently precise, but each method recovered different genes known to be required for viability (Morgens et al...list emerge that accurately reflected the scope of genes known to be essential for cell growth. Together,...

...predicted genes have unidentified roles as well (Vanni, et al). Is it true that this many genes are truly...biologically relevant situations. Tissue-specific genes – the genes of interest may not be expressed in the tissue... be observed. Functionally redundant genes – multiple genes which function in overlapping roles can...Around 20% of human genes have no known function or a poorly defined function (Wood, et al). In the microbial...line you are working with. Development-specific genes – if a gene functions at multiple stages in development...the organism you are working in. The spacing of genes and whether or not it’s ok to disrupt endogenous...construct to generate a collection of monitored genes and mutants - viral integration, transposon, and...

... one bacterium sharing genes with another, an accidental transmission of genes from one bacterium to another...without a new, drug-resistant ‘superbug’ making headlines in one part of the world or another. Bacterial...part in our own health and the health of our loved ones, and become a positive force for public health. ...consuming to treat. Learn How Antibiotic Resistance Genes Are Used in the Lab How does antibiotic resistance...bacterial cells), or a bacterium picking up resistance genes from it’s environment (from DNA left behind after...while the few that happen to possess resistance genes survive and multiply. Factors contributing to the... bacteria are resistant to antibiotics and which ones aren't?     This video shows how to perform a...

...research for single or multiple disciplines – you can select the disciplines and the years. Which leads to... as researchers in other countries or ot­her disciplines, are even less likely to read about our work.... have time to read about research from other disciplines, or from other countries, to explore new connections...the challenge of keeping up on studies in many disciplines has been a long brewing frustration for me. I... I read a variety of psychology, business, personnel, and sociology journals when I get a chance, but ...from ideas garnered from research in another disciplines – maybe psychology, maybe economics, or maybe...increasingly citing research from outside of their disciplines. Included in the article is an interactive chart...

...viscosus, Aerobacter aerogenes, Aeromonas hydrophila, Agrobacterium tumefaciens, Alcaligenes eutrophus, Azotobacter...salmonicida, Agrobacterium tumefaciens, A. rhizogenes, Alcaligenes eutrophus, Enterobacter sp., Erwinia amylovora...plasmids in nature, and provide tips on how we can harness those features to create broad host range recombinant...range and can replicate and stably maintain the genes they carry among organisms belonging to many different...processes, and can be used by scientists across disciplines.  What confers broad host range in plasmids? ...that the plasmid is conjugative and carries the genes necessary for transfer initiation between bacterial...Acinetobacter spp., Aeromonas spp., Agrobacterium spp., Alcaligenes spp., Aliivibrio salmonicida, Anabaena spp., ...

...permissive cell lines such as HEK293, H0 or A549 and assume that the results obtained in these lines can be translated...laboratory tool often employed to establish cell lines that stably express a gene of interest. While the...lead to low titers and poor transductions. Cell lines should be routinely subcultured at densities that... cells do not take up nucleic acid or express transgenes as well as actively dividing cells resulting ...personnel. If you are unsure of the background of the lines you are using, it is best to start from scratch.... a variety of sources such as serum, other cell lines, or infected personnel and can persist undetected...mycoplasma detection kits that use detector cell lines, ELISA, fluorometry, or PCR-based approaches to ...

...the mRNA vaccines While there have been many studies on the effectiveness of current vaccines against ...lab found that the neutralization effectiveness of current vaccines differed depending on the variants...vaccination efforts. So, how effective are the current vaccines against the variants? Alejandro Balazs, a Group...assay. Neutralization was quantified in terms of luminescence. Image from Garcia-Beltran et al., 2021 with...key drivers in determining whether the current vaccines are effective or not. This protein both elicits...elicits an immune response as the target of vaccines and allows the virus to gain entry into human cells via...target cells and this can be detected via loss of luminescence.     B.1.351 variant neutralization escape ...

...using two membranes during your transfer instead of one. If you see signal on both membranes, you know... run the same protein concentration in multiple lanes and cut up the membrane to treat each lane with ...   Figure 2: Reagent gradient. Here, the lanes are lightly marked on the membrane (1) using the... time.   Samples can run into empty neighboring lanes, so load your gel to ensure your samples and reference...troubleshoot.   Before you start… Mark your membrane! Membranes look the same from both sides, so it’s quite easy...may be under-transferring.  Membrane pore size Membranes come in different pore sizes for different proteins...(< 15 kDa) and a 0.45 µm (p) membrane for larger ones. Changing your membrane size is probably the easiest...

...repression of target genes in plants and many of the aforementioned empty gRNA backbones also contain Cas9...for targeted modification of known or suspected genes that regulate a desired phenotype. In fact, CRISPR...be used to knockout, activate or repress target genes in plants using the same general experimental design...cells. Like other model systems, expression of S. pyogenes Cas9 or Cas9 variants (hereafter referred to as...respective species. Addgene carries >30 “empty gRNA” backbones which contain a plant pol III promoter and gRNA... can also be used to activate or repress target genes in plant cells, respectively. Cas9 expression is..., and a Ti "helper" plasmid, which contains Vir genes necessary for T-DNA processing and insertion into...

...individual clones from that population. You can additionally calculate the minimum number of clones you would... sequence clones to verify that the deletions introduced are out of frame but finding clones with deletions... success? Before you start analyzing individual clones, it’s wise to quickly assess whether a significant...verify that you won’t need to screen hundreds of clones looking for your edit of interest. For all of the...before going on to generate and screen individual clones. If a clonal line isn’t your end goal, then a single...is sequencing trace files of WT and edited cell lines. Then decomposition analysis is performed yielding... clone, you should screen, at the very least, 4 clones. Did you use a dual guide system to generate your...

... by the heavy-chain constant genes μ and ẟ. These are the first two genes in the heavy-chain constant ...intervening heavy-chain constant domain genes, placing the variable domain genes next to a different constant domain... you heard of an antibody isotype? An isotype determines several key characteristics of an antibody as...an antibody’s isotype is important because it determines what immune cells and molecules are recruited...nomenclature. Antibody heavy chain proteins as well as the genes that encode those proteins are designated by the...tears, saliva, mucus) where it protects mucosal membranes. This isotype is most commonly a dimer with a ...type of DNA recombination. Heavy chain constant genes are clustered together downstream of the variable...

...planarian genes, we encourage you to deposit them back with Addgene and expand the number of genes that other...Hybridization (WISH) for visualizing expression of genes. Double-stranded RNA (dsRNA)-mediated RNA interference...expression and functions of key regeneration-related genes in four inexpensive and commercially available planarians...and Phagocata gracillis. There are 11 different genes cloned into plasmids to investigate for each of ...changes to visualize the expression of specific genes. After riboprobe binding (via hybridization) specific...The expression patterns of 11 different planarian genes (all of which are available through the deposit)...powerful tool for performing targeted knockdowns of genes of interest and was first discovered and characterized...

...computational methods to identify genes by their similarities to genes in other species, but these studies...MET genes to grow.) Students then use replica plating to determine if expression of MET/Met genes from... to answer questions about the evolution of the genes involved in methionine synthesis (5). The budding...strains with overexpression plasmids carrying MET/Met genes from either S. pombe or S. cerevisiae. Students ... the coding sequences for ten different MET/Met genes (Figure 1) from the two yeast species into the pYES2.1...uracil supplementation (6). Expression of MET/Met genes from the pYES2.1 plasmid is controlled by the GAL1...have shown that most, but not all, S. pombe Met genes complement the corresponding S. cerevisiae met deficiencies...

... light include chemiluminescent proteins, like luciferases, as well as fluorescent ones, like Green Fluorescent...that, although they are not bioluminescent, some types of corals and anemones are vibrantly colored. He ...Luminescent molecules are very useful tools because we can easily detect and measure the light they emit...Protein (GFP). These molecules occur naturally in bioluminescent organisms, but their real power lies in the...interesting. Although humans have known about bioluminescent organisms and their characteristic radiance..., in the 1960s, Osamu Shimomura studied the bioluminescent properties observed in the crystal jellyfish...the culprits. He even obtained samples of certain ones from local pet shop aquariums in Moscow where he...