...vitro as well as in vivo in yeast, plant, insect, and mammalian cells, including human cells. piggyBac...is active in vertebrates and integrates in human cells at rates similar to retroviral vectors. The hyperactive...piggyBac has an integration efficiency in human cells similar to retroviral vectors. The hyperactive PB...transposase (hyPB) is ~10-fold more active in mammalian cells compared to the codon-optimized wild-type piggyBac...G/C)TNA. Tol2 can deliver 10-11 kB to mammalian cells without decreased efficiency, with a maximum cargo...and has lower efficiency of integration in human cells than piggyBac and Sleeping Beauty. Minimal Tol2 ... or barcodes. When transposons are delivered to cells or a model organism, they insert into the host’s...

...For about as long as the disciplines have existed, cell and molecular biologists have been continuously ... peer into the mysterious, microscopic world of cells. The development of antibodies as tools allowed ...was the existence of high quality antibodies for well defined peptides. Munro and Pelham’s first published...monoclonals would help the field explore Myc’s role in cell proliferation and tumorigenesis.  Using two different...the same antibody developed almost 40 years ago? Well…kinda. In some cases, antibody providers are distributing... human IgG2, mouse IgE, goat IgG, (and more!) as well as the original mouse IgG1. And the refinement of...specific for human c-myc proto-oncogene product. Mol Cell Biol 5:3610–3616. https://doi.org/10.1128/mcb.5.12.3610...

...check its subcellular location and solubility. If it’s membrane-bound or located in a subcellular compartment...reagent. Be aware that this approach requires excellent attention to detail, since you’ll end up with ...question before running one.  Good on gradients? Excellent! Now, let’s head west, intrepid reader! Protein...strengths. If that’s not working, consider using a subcellular fractionation kit or a lysing protocol developed...samples and reference ladders are not next to empty wells. Most people will simply load an extra ladder or...replicate (ie, leftover sample) to fill the relevant wells. Similarly, if you load a gel and then forget to...the time with smaller tasks, or simply enjoy some well-deserved down time while waiting for your timer ...

...both SpCas9 and your gRNA in a variety of mammalian cell types. Purpose  SpCas9 This plasmid expresses Streptococcus...the Zhang lab has tested in a variety of mammalian cell lines. Cas9, of course, is the key component of ... puromycin resistance gene enables selection of cells with the plasmid post transfection. The 2A "self...can be used to transfect a variety of mammalian cells and can be modified for viral trasduction (see the...resistance gene that made it less effective in certain cell lines. This point mutation has been corrected in... your gRNA of interest in a variety of mammalian cell lines. Alternative forms of this vector contain ...directed repair. You can find all of these variants, as well as links to the Zhang Lab's Nature Protocols Publication...

...scientists. What works well in one cell or neuron type may not work well for a neighboring cell type, even within...options while AAV8 (82%) and AAV9 (84%) were both excellent at infecting our target neurons (Figure 1). However... conditions (3,5,8), injection titers (1,2), and cell types used for packaging viral particles (6), can...methods and image descriptions, a minimum n = 2, and well labeled high- and low-magnification images demonstrating... vector performance in various brain regions and cell types. Power in numbers A Pubmed search for “AAV....169 Gruntman, A. M., Su, L., Su, Q., Gao, G., Mueller, C., & Flotte, T. R. (2015). Stability and compatibility...

...Class 2 CRISPR-Cas System. Cell 163:759–771 . https://doi.org/10.1016/j.cell.2015.09.038 Find plasmids...are capable of cleaving genomic DNA in mammalian cells and zebrafish embryos, and they can be used to modify...domains and allows for expanded PAM recognition as well as increased specificity and lower off-target activity... adapted for targeted RNA cleavage in mammalian cells. The REPAIR (RNA Editing for Programmable A to I...Han S, Joung J, Belanto JJ, Verdine V, Cox DBT, Kellner MJ, Regev A, Lander ES, Voytas DF, Ting AY, Zhang... DBT, Gootenberg JS, Abudayyeh OO, Franklin B, Kellner MJ, Joung J, Zhang F (2017) RNA editing with CRISPR-Cas13...reveals specificities of Cpf1 endonucleases in human cells. Nat Biotechnol 34:863–868 . https://doi.org/10.1038...

...blog The blog has many other excellent posts on science career topics as well. Instead of rewriting or coming...everyone, particularly if you’re more interested in selling a product than communicating your ideas with people...has great questions! 2. The r/Science subreddit is well moderated. One of our original worries about sharing...rousing the trolls. 3. The AMA process was simple and well organized. You can check out the r/Science AMA Submission...with our general mission to share- as opposed to sell- science. 4. You can prepare answers beforehand....answers off the cuff, but like to be able to provide well thought out responses. I hung out with Joanne as...small quibble. If you are not too worried about selling yourself and really do just want to get information...

... small circular piece of DNA found in bacterial cells, and someone new to plasmids may need some extra...and designed to introduce foreign DNA into another cell. Minimally, lab-created plasmids have an origin ...the ability of plasmids to self-replicate within a cell make them attractive tools for the life scientist...component for expression vectors: determines which cell types the gene is expressed in and amount of recombinant...plasmids also have selectable markers for use in other cell types. Primer Binding Site A short single-stranded...newly inserted gene is transformed into bacterial cells and selectively grown on antibiotic plates.  Importantly...plasmids to manipulate gene expression in target cells. Characteristics such as flexibility, versatility...

...aspect of being a successful manager. Great bosses tell people where they stand clearly and routinely. They... are clear with each employee about what they do well and where they need to improve, and they’re also...making it a concrete goal to say “thank you, job well done” to one of your employees every day. Prepare... is usually good. Don’t pretend something went well when it didn’t… but don’t “punish” someone for a...say worked out best? What 3 things worked least well? Manager provides feedback (may be the same as ...best.  Here are the 3 things that worked least well. Find out the Effect of the Feedback: How does...this feedback compare with your own perception? Tell me your interpretation of my feedback to you. What...

... details for the march in Washington as well as the satellite marches. Anyone can join the marches or ... by Addgene or its staff. In Washington, D.C. as well as sister locations on April 22, 2017, scientists...government protects (and often funds [3]) scientists as well as other citizens. Thus civilization proceeds in...lists over 200 partner organizations, and 514 satellite marches are scheduled to take place around the... a success. Below are resources for the march as well as other science advocacy opportunities. Information...conversation about science’s role in government as well as active advocacy will be required. Information...Website The Washington Post: Trump administration tells EPA to freeze all grants, contracts The Guardian...

... that converts sucrose into a toxic metabolite. Cells that have undergone a double crossover will not ...contain sacB, allowing them to grow on sucrose. Cells that have only gone through the single cross over...counter-selection escape). The rhamnose induction system is well-characterized and quite tight (Giacalone et al.,...., 2006). The Chen lab demonstrated excellent counter-selection which enabled very efficient recombineering...desired deletion directly into cut vector with excellent efficiency using a commercial isothermal assembly...Enterobacteriaceae including in E. coli O157:H7, Shigella flexneri, Enterobacter cloacae, and Serratia marcescens...marcescens and have created dozens of deletions as well as insertions with this system (Lazarus et al., ...

...read some of the seminal papers on cell cycle discovery from Lee Hartwell and Paul Nurse,” Stegmeier says...regulate cell division. And that really got me hooked on genetics.” As a postdoc in Stephen Elledge at Harvard...a great chemistry teacher in 10th grade and then fell in love with science,” she says. In 11th grade she...high-resolution tracking of more than 1 million cancer cells under drug treatment.” Stegmeier especially finds...Belgium. “That’s where I not only got incredibly compelled by wet bench molecular research, but also became...these inefficiencies is pretty remarkable. I was compelled to be part of that.” He’s deposited AAV constructs...

...imaging with single-cell resolution using chemical cocktails and computational analysis." Cell 157.3 (2014):... of the XFP genes is introduced at ~2 copies per cell per color, following a Poisson distribution. To ... optimal expression of XFPs. Any longer and the cells may start to show morphological abnormalities due...XFP genes have to be introduced at ~2 copies per cell per color to achieve the highest color variations... wait too long, as too much XFP is toxic to the cells. Inadequate expression levels of XFPs - Reduce the...video below depicting dendrite wiring of mitral cells.   Many thanks to our guest bloggers Richi Sakaguchi...circuitry with an index-optimized clearing agent." Cell reports14.11 (2016): 2718-2732. PubMed PMID: 26972009...

... there is one problem: how to get that into the cells of interest,” Griesbeck said. Because they are chemical...understand the connection between molecular- and cellular-level events and behavior. It’s a problem that...advantage for observing events taking place inside cells that are on the move, such as T lymphocytes. Griesbeck...-binding protein, troponin C (TnC), from muscle cells. They identified a toadfish TnC variant which had...cAMP-responsive element-binding protein (CREB) in living cells. “Now we are making this really good,” Griesbeck...FRET-based sensor proteins, particularly for the intracellular imaging of transition metal ions, such as zinc...also present challenges in that they are found in cells at much lower concentrations than calcium, requiring...

...recently re-amplified our distribution stock of the Brunello Human gRNA library, and we thought it would be... Typically, Addgene uses electrocompetent Stbl4 cells when performing amplifications, and we use electroporation...DNA. This bias can be counteracted by growing the cells on solid plates, where each transformant forms its...own colony, uninfluenced by the growth of other cells. For libraries where the plasmids are all very similar...The colonies are then scraped off the plates and pelleted for DNA extraction. Prepare library product for...PCR. A diagram of the product we created for the Brunello library is shown in Figure 2. You can use a small...plotted as a histogram, the counts will give you a bell curve of the abundance of each component of your...

...at lower copy numbers within bacterial cells, and bacterial cells with multimers grow more slowly compared...remain intact, plasmid multimers function just as well as monomers. However, due to their larger size, ...stable and more prone to being lost in bacterial cells compared to monomers, although this can be prevented...increase the frequency of base substitution in human cells. J Biol Chem. 2011 Mar 25;286(12):10017-26. doi:...plasmids leading to the formation of plasmid multimers. Cell. 1976 Dec;9(4 PT 2):707-16. doi: 10.1016/0092-8674...supercoiled plasmid DNA concatemers in mammalian cells. J Gene Med. 2009 May;11(5):444-53. doi: 10.1002...essential for plasmid monomerization and stability. Cell. 1984 Apr;36(4):1097-103. doi: 10.1016/0092-8674...

...gives a talk at the Cell Symposia: Gene- and Cell-based Therapies: CRISPR, Stem Cells, and Beyond meeting...contains the deoxyadenosine deaminase, TadA-7.10 (Gaudelli et al., 2017). The idea was to evolve a TadA-7.10...III product is produced and can infect bacterial cells. If a phage does not deliver an active variant, ...passage from the infected to a fresh uninfected host cell culture). This dilution step represents the major...but gets manually passaged from an infected host-cell culture to an uninfected culture which results in...efficiently and in more contexts. References Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson...

...cryopreserved hybridomas, immortalized cells created by fusing an activated B cell that produces the antibody of...of interest with a myeloma cell. This RNA was then used to generate cDNA, which  was used as a template...of hybridoma generated mAbs is that if the frozen cell line becomes non-recoverable, the stock of the mAb...hybridoma stocks, without having to grow up the cells. This RNA could then be used to generate functional...immunofluorescence-immunocytochemistry (IF-ICC.) This method was chosen because cells transfected with a plasmid to express the target... could be screened in a high-throughput manner. Cells were labeled with both a hybridoma generated mAb...hybridoma mAb.  While screening by IF-ICC worked well for identification of the correct clones, it was...

...Translocations in Human Cells Are Generated by Canonical Nonhomologous End-Joining. Molecular Cell 55:829–842 . ...Genome Editing Specificity. Cell 154:1380–1389 . https://doi.org/10.1016/j.cell.2013.08.021 Waters CA, Strande... chromosomal DSBs. It is important to know that cells possess two major repair pathways – Non-Homologous... steps Unlike HDR, NHEJ is active throughout the cell cycle and has a higher capacity for repair, as there...should take less than an hour, thus a population of cells constitutively expressing a targeted Cas9 should...CRISPR Protocol for Genomic Deletions in Mammalian Cell Lines [Video] Pooled CRISPR Libraries Offer Genome-Wide...

...proteins in cells. Homeostatic regulation of protein abundance is essential for normal cell function, and...one particular protein in neurons or in any other cell type. However, we cannot distinguish new from old...facilitate the visualization of specific proteins in cells. Tagged protein can easily be overexpressed from...techniques to follow translation in living single cells. However, before CRISPR-Cas9 these techniques were...interests, suspected to interact, are added to a cellular sample. Both antibodies have a short antibody ... turnover of specific endogenous proteins in any cell type. Exciting! Many thanks to our guest blogger... how small molecule fluorophores can be used for cell labeling Check out other protein tagging strategies...