...of interest as well as sequences necessary to encapsulate that insert into a viral particle (the packaging...lentiviral vector system is the tetracycline (Tet)-regulated system. You can choose either the Tet-off system...rtTA (FUW-M2rtTA). In this kind of approach, a population of cells might express only the tTA or your transgene...proto-oncogenes or oncogenes for instance should be manipulated with care. See the NIH’s biosafety guidelines...viral capsid antigen p24, the FACS dosing method correlates viral titre to the number of cells transduced...order to aid viral binding. This method, called spinoculation, has been found very effective for T cells transduction...Yasutsugu Suzuki and Youichi Suzuki (2011). Gene Regulatable Lentiviral Vector System, Viral Gene Therapy,...

...technology (Yesbolatova et al., 2019). Find the mAID tagging toolkit at Addgene Yesbolatova et al., Methods...ubiquitin ligase, the Kanemaki lab was able to translate AID to yeast, as well as mammalian and human cell...tagging of proteins with mAID and other tags (Yesbolatova et al., 2019) using CRISPR. This toolkit makes...thought to play a key role in the transcriptional regulation of many genes. A number of groups have designed...synthetic looping factors developed thus far for these latter experiments, such as transcription factors tethered...

...genetic manipulation more hands on While teaching courses about gene therapy and genetic manipulation, I and...on genetic manipulation of human stem cells when students are not even allowed to manipulate prokaryotic...transfer plasmid that could be used to genetically manipulate human stem cells. Then they had 10 minutes to...explain why they selected a certain target cell population. Who is the winner? Ultimately, society should...

...Kurt Thorn (UCSF at the time) began an effort to collate some of the current “best” FPs, and I collaborated...using simple web forms) while also maintaining a relatively strict database design (schema).  Registered ...allows for visual comparison of FPs and how they relate to one another. .  In addition, the FPbase Fluorescence...feature filter spectra and collection efficiency calculations.  With ~80 FP spectra (and rapidly growing, ... page with all the information in the database relating to that specific protein (for instance: EGFP)....retrieve all of the information in the database relating to that set of proteins.  For computer-savvy users...

...might not have to do the calculation yourself as many fluorometers will calculate your sample concentration...than 1.5. Then, using the A260 reading, you can calculate the DNA concentration. Generally, A260 of 1.0 ... start by pouring your gel containing a DNA intercalating dye (ex: ethidium bromide) and choosing a DNA...quantify based on band intensities of your DNA relative to the intensity in the ladder. It’s best to compare...quantified over time using a fluorescent dye that intercalates into the DNA such that smaller fragments are...

...Watch out for methylation issues. Enzymes like XbaI and ClaI are sensitive to methylation and their activity... Congratulations, you have a plasmid expressing your gene of interest (YGOI) and are ready to dive into...the reaction on an agarose gel to determine the relative sizes of the resulting DNA fragments. Restriction...will need to purify your DNA from a dcm or dam methylation-deficient bacterial strain such as JM110 or INV110...enzymes are working. When uncut plasmid DNA is isolated and run on an agarose gel, you are likely to see...

... Sensitivity Cas9 sensitivity is often closely related to specificity. Sensitivity is a measure of the...site. A specificity ratio can be measured by calculating the on-target activity divided by the off-target... SpCas9 described the PAM specificity of Cas9s isolated from different organisms and engineered Cas9 variants...the enzymes showed decreased off-target activity relative to Cas9. Since then, the Joung and Jennifer Doudna...showed decreased or similar off-target activity, relative to eSpCas9 and SpCas9-HF across a number of different...research due to its small size and specificity relative to Cas9. As scientists continue to characterize...

...devices for research. There are a number of new platforms (such as openUC2, which I am a co-founder of),...that make everyday microscopy much easier. On platforms such as Github, users can download the software...example, can also be printed quickly in Madagascar. Platforms that allow for on-going creativity with the construction...organizations and companies to provide problem-solving platforms.     Fig. 3: Different workflows involved...their work on the shoulders of giants. The still relatively young movement of sharing open hardware has a...Prakash, "Squid: Simplifying Quantitative Imaging Platform Development and Deployment." bioRxiv. 2020. Diederich...

...limited to: rapid growth, ease of replica plating and mutant isolation, a well-defined genetic system, and ...and animals. Unlike bacteria, yeast can post-translationally modify proteins yet they still share many of...a previous post, we briefly discussed how the regulation of bacterial ORIs determines plasmid copy number...these vectors can also be controlled if specific regulatable elements are included (reviewed here). Plasmids...complementation is weak. yes   LYS2 L-lysine yes - Grow on plates containing alpha-aminoadipate in the absence of...

...brightness for a few commonly used fluorophores in relation to the laser type. Table adapted from: https:/...are absorbed and then emitted, which creates a relatively stronger — or brighter — signal.   Figure...use brighter fluorophores for markers that are relatively lower in abundance. In contrast, when you expect...the spectra of enhanced GFP (eGFP) and FITC, the latter being a commonly used fluorophore for antibody-...again. In the beginning, we talked about the relationship between a photon's wavelength and energy, how... into different levels of brightness and their relation to the fluorophore's excitation efficiency, and.... His projects include investigating negative regulators of T cells and cytokine signaling in tumor immunity...

...future prospects. Among the topics discussed, many related to the structure of the biomedical workforce. Quotes...results, not scientists”  These comments are well correlated with published data. For example, in 2010, less...compulsory internship to be taken in an industry, regulatory, or clinical environment. It remains contested...6). Academic data and the labor market for PhDs Later this week, postdocs and students will again gather...grants through the Sloan foundation for postdoc-related data collection and analysis. He is also a strong...training to reflect the workforce." Science Translational Medicine (2015): 285ED4. Pubmed PMID: 25925677...

...is the question. When presenting on building relationships (also known as “networking”), one of the most...networking to start, build, and track professional relationships. Here are my seven best tips beyond the basics... “Joanne Kamens, PhD viewed your profile.”  The latter is much less creepy. If we’ve met before, I’d be...on LinkedIn!” resulting in just a little more relationship. Pro tip: Once you are connected with someone...connection requests LinkedIn is a place to build relationships. If you don’t add a personal note, you waste...volunteer organizational work. LinkedIn is not the platform for politics, family updates, or click bait of...

...specific, individual proteins under endogenous regulatory control. But even with the advent of CRISPR/Cas9...the gene to ensure it would be transcribed and translated. For an N-terminally tagged protein, we used ...human genes and will add more to the collection later this year. Designing the donor plasmid A key feature... one thousand base pairs on each side, in fact. Later, when we transfect the cells, these large regions...percent of cases, the stem cell will seek out a template for the repair process. By flooding the cell with...using the introduced donor plasmid as a repair template. This results in the insertion of the GFP tag ...

...Unfortunately, plasmid copy numbers vary within cell populations and over time resulting in variable gene expression... use of stabilized promoters. In contrast to unregulated promoters that are always on, stabilized promoters...expression. However, if you introduce negative regulation by using a promoter that is responsive to a repressor...that the stabilized promoters were able to better modulate gene expression during shifts in growth rate as... as compared to a constitutive promoter. To simulate the increase in plasmid copy number during the transition...generation of the desired product while avoiding the accumulation of toxic intermediates or the shuttling of the...

...purification. In this strategy a single protein can be isolated from a complex mix using affinity chromatography...variety of methods can be used to successfully isolate proteins, this blog will focus on one of the most...the protein of interest and it is successfully isolated.   Prior to the polyhistidine tag, alternative...binding of untagged proteins. Despite being a relatively low frequency amino acid, proteins containing...questions about polyhistidine tags. For more antibody-related information, sign up to receive alerts for antibody...Purification of Recombinant Proteins with a Novel Metal Chelate Adsorbent. Bio/Technology. 1988; 6: 1321–1325. ...

...have adapted the components for use in genome manipulation. Cre recombinase, originally named because ...because it “causes recombination” (although later referred to as the “cyclization recombinase”), is a 38 kDa... in a DNA sequence allows for very specific manipulations as discussed below. How does it work? As alluded...expression – placing a series of transcriptional and translational termination features with loxP sites on either... can be used to easily perform this eviction. Regulated Cre expression – placing Cre downstream of promoters...express Cre under a variety of ubiquitous and regulated promoters, and many loxP-containing alleles have...

...City The conference was located in the modern, relatively new “Science City” area on the outskirts of Guangzhouon...human tRNA genes and, until now, we've overlooked regulatory fragments of these transcripts. I remember when...have allowed them to begin understanding complex regulation of and by tRNAs and tRNA-derived RNAs. Stay tuned...Medicine presented another unexpected finding in the regulatory RNA field. The Rajewsky lab is studying circular...of Georgia studies the CRISPR/Cas system as it relates to its natural role in prokaryotic immunity to ...against both DNA and RNA viruses - 2 cas systems regulate at the DNA level and 1 at the RNA level. Udi Qimron...

...field as it provides a noninvasive method for manipulating cell activity, especially neuronal firing. ...approved drugs so that your results are viable for translation to human patients. Picking receptors The receptor...dependent on what cellular response you aim to manipulate. It is important to consider that the same receptor... in. The most common target for chemogenetic manipulation are neurons where chemogenetic receptors can...control ion flux. The system allows you to directly manipulate ion channels. When working with PSAM receptors...BL, Jin J (2015) The First Structure–Activity Relationship Studies for Designer Receptors Exclusively Activated...

...calls, I feel compelled to share my years of accumulated wisdom so that you, the customer, can get the...these tips and tricks will apply to any biology-related customer support interaction. Before contacting...paperwork associated with a product only to realize later that everything I could have wanted to know was ... science, but we don’t have the resources to recapitulate the experiments performed by our thousands of...use a particular reagent.  We also have links to later papers that successfully used said plasmids. We ...support scientists wear many hats at this still relatively small nonprofit. If you do go longer than a few...

...received over 13,000 plasmids requests for COVID-19 related plasmids. These plasmids have reached labs in over...expression and pseudotyping efficiency. This plasmid was later used by another team of researchers for neutralization...opportunities to evolve. Several variants are now circulating and plasmids containing these mutations have ...are inhibitors of SARS-CoV-2 entry and become upregulated during viral infection.  Expression of SARS-CoV...their work, head over to their blog post. Months later, we partnered with Ginkgo Bioworks to provide synthesized...screens identify mucins as critical host factors modulating SARS-CoV-2 infection. bioRxiv. https://doi.org...