...benefits commensurate with work training and experience, better align trainee expectations with job outcomes...that fact that training in the academic research environment is setup to prepare trainees for further work...non-academic careers, much of the training they receive as postdocs is not transferrable to other environments. ... of trainees at low wages will still be there without restructuring. Positive Points for Trainees - Taking...information - all traits you are likely to find in graduate students and postdocs. Trainees however, need ... the mentor and this training is clearly not equitable to that of other trainees despite the fact that...temporary and defined period of mentored advanced training to enhance the professional skills and research...

...Viral delivery is attractive due to the ease of transduction in difficult-to-transfect cell types ranging...higher than traditional transfection and random genomic integration of a plasmid. Viral transduction is also...overexpression of all human transcription factors, barcoded libraries to trace cell lineage, and so much...delivered with viral vectors instead of a traditional transfection method. This is because viral systems ...integrated into the genome of the target cell or transiently expressed. It’s important to note that for all...vector can express your construct in target cells transiently or constitutively depending on the virus used...also generally less toxic to cells than transfections, preserving cell viability. Library delivery Screening...

...droplets is used to calculate the concentration of the sample. This concentration can then be used to calculate...possible to measure the concentration of multiple targets simultaneously. For AAV titration, you should only ...tool for quantifying AAV for some time now. When titrating AAV by qPCR, viral DNA is amplified and monitored...For these reasons, we are in the process of transitioning our titering method from qPCR to ddPCR. Droplet...needed for your PCR experiment. The process to titrate AAV by ddPCR begins with diluting the virus. It...million.  After making the dilutions, they are transferred to a new plate containing the mastermix which...place.  When the PCR is complete, a droplet reader extracts the droplets from the plate and measures the fluorescence...

...energy transfer) and FLIM (Fluorescence Lifetime Imaging Microscopy) applications. To demonstrate the power...caging technique to beautifully demonstrate light-dependent Gal4 transcriptional activation and Cre recombination...scientists to quickly and easily generate zebrafish transgenic lines to study neurobiology and neuorodgenerative... six fluorophores with nonoverlapping emission spectra (N- and C-terminal tags for mTagBFP, TagRFPt, EGFP...pluripotent stem cells) through the expression of transcription factors OCT3/4, SOX2, KLF4, and c-MYC is the... method for generating iPSCs to express the transcription factors from a polycistronic cassette. However...comparable experimental results. Using PiggyBac transposons, the Woltjen Lab compared different variants ...

... Wenning Qin. Single-stranded DNA repair templates Short single-stranded DNA templates have been shown...HDR subpathway. It's traditionally been difficult to produce long single-stranded DNA, but Miura et al...perform in vitro transcription, and use the IVT product as a template for reverse transcription to create your...order to insert two loxP sites. Since two double-strand breaks are made, it is common to see deletions ...structure. Today, multiple companies offer long single-stranded DNA production services or kits, including the... the separated guide RNA components (crRNA and tracrRNA) for maximum Easi-CRISPR efficiency. Easi-CRISPR...microinjection and electroporation methods using in mouse transgenics. Designing an Easi-CRISPR knock-in allele To ...

...via the Sec translocon." Traffic 12.5 (2011): 543-548. Pubmed PMID: 21255213. Pubmed Central PMCID: PMC3079558...[3]. You must therefore determine your cloning strategy and decide whether you will create an N- or C-...mitochondria, lysosomes or they are secreted into the extracellular milieu. Many of the resident proteins within...these compartments undergo significant post-translational modifications including glycosylation, disulfide... addition of N-glycans [9]. Both of these posttranslational modifications can disrupt FP folding rendering...and yellow moxFPs. The inherent brightness and spectral characteristics of the moxFPs are comparable to...non-optimized parental proteins. Our recent publication illustrated the quantitative increase in fluorescent signal...

...changes in intracellular Ca2+ levels than the original version containing CRY2 without the extra peptide....-free drinks. Finally, they demonstrated that the caffeine concentrations in several beverages could be...networks. A major drawback of this method is the stray excitation of neighboring neurons in opsin dense... (Figure 1.) Moreover, the group was able to demonstrate that while illumination of neighboring cells ...expression in E. coli. This operon is under the transcriptional control of a strong constitutive promoter (... required enzymes, including a glutathione S-transferase homolog from a Janthinobacterium sp. Moreover...Moreover, a caffeine “addicted” E. coli strain expressing this operon can serve as a living biosensor for measuring...

...its ability to infiltrate cells. Options for CRISPR delivery include plasmid transfection, RNP electroporation...been used to increase retroviral transduction efficiency by neutralizing the charge repulsion between virions...results demonstrate that Nanoblades are a potential alternative method for generating transgenic mice strains...electroporation, and viral transduction; but these methods aren’t stealthy enough to gain access to some cells...Repair (HDR), CRISPR activation or repression, transgenic mouse line creation, and in vivo gene editing... non-replicative. Nanoblades are produced by transfecting the following four components into 293T cells... can also deliver Cas9 variants, such as a transcriptional activator dCas9-VPR, if these variants are ...

...Pick the right strain While common E. coli strains like DH5-alpha are recA-, some strains have been engineered...suggest gel extraction to isolate the full, intact plasmid. Please see our gel extraction protocol for...Learn about the common lab E. coli strains Find tips for viral transduction  ...Repeating sequences within a plasmid can undergo intramolecular DNA recombination. This naturally occurring...recombination, one resulting plasmid carries the transgene of interest (aka the viral genome) and the other...resistance gene and origin will replicate, and the transgene will be lost. Over time, bacteria containing the...further reduce recombination. Recombinase-deficient strains like Stbl2, Stbl3, and NEB stable E. coli reduce...

...biofactories by demonstrating the use of CRISPR-based RNA scaffolds to mimic natural transcriptional programs...recent paper, "Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds." Adam has...visualize the concepts discussed in the paper. The transcriptional control of multiple loci is deftly coordinated...cerevisiae with few techniques that are easily transferrable across host species or metabolic pathways. A...synthetic RNA scaffolds that can recruit various transcriptional regulators (Zalatan et al. 2015).  These scaffolds...nucleotide sequence then subsequently recruit a transcriptional regulator to modulate expression at that locus...expression levels dependent upon the type of transcriptional regulator or the number of designed scaffold...

...activity and instead become trapped in a covalent bond with its substrate. Then, all it takes is a bit...range of useful molecules linked to that substrate. If the substrate was linked to, say, a chemical dye or...applications.   Figure 1: Live-cell labeling strategies. The green structure represents a fluorophore...a look at some popular self-labeling tools: Tetracysteine: The first reported chemical labeling tag, from... gets covalently linked to derivatives of its substrate O6-benzylguanine (BG) (Keppler et al., 2003). ...The Virginia Cornish and Michael Sheetz labs demonstrated in 2005 that proteins fused with E. coli dihydrofolate...be labeled with fluorescent derivatives of its substrate trimpethoprim (TMP-tag) (Miller et al., 2005)....

...your strategy. I realized that similar types of thinking go into developing gene therapy strategies for...logic and strategy skills to apply those concepts and understand how therapeutic strategies work. Find...game. They had 35 minutes to develop a strategy and build a transfer plasmid that could be used to genetically... a win-win strategy. After each team presented, all students had to pick the best strategy and propose... This guest post was contributed by Marco Straccia, an Associate Professor at University of Barcelona...the University of Barcelona wanted to develop strategies to make our classes more practical and hands-...biology, microbiology, genetic engineering and strategic knowledge to solve a clinical disease case. As...

...:     Cells must be amenable to transfection or viral transduction     Appropriate promoters must be...expression     The requirement for Cas9 transcription and translation delays editing What are Cas9-gRNA ... studies in cells that are difficult to transfect or transduce. While using Cas9-gRNA RNPs may require...site of interest. This is commonly performed by transfecting cells with a plasmid, such as PX459, which encodes... often be used in cells that are difficult to transfect, such as primary cells. Using RNPs can also alleviate... are detectable at high levels shortly after transfection, and are quickly cleared from the cell via protein...from Alex Schier’s Lab. gRNAs can be in vitro transcribed from ssDNA, which can be generated by commercial...

...Types of transfers Your transfer options are wet, semi-dry, or dry/electroblot. A wet transfer is the most... while semi-dry transfers require a semi-dry transfer cell. Dry/electroblot transfers only work in the...) steps to the transfer: you’ll need to pre-wet most PVDF membranes before the transfer and allow them...  Note that each type of transfer requires different equipment. Wet transfers can be done in an insert...strip/reprobe Semi-dry transfer Decreased efficiency Electroblot transfer Increased cost; decreased...strong as DTT, you’ll have to use different concentrations if you switch from one to the other.  Type ... 180 V, 30 minutes Good shelf life; runs at neutral pH (reduced protein alteration) Expensive buffers...

...through transformation, so the cloned vector must be ligated. But in LIC, longer single-stranded overhangs...higher DNA concentrations are needed for this method to be successful. Low DNA concentration? No problem...overlapping regions of single-stranded DNA. But as long as the double-stranded region is long enough to hold...LIC) was first developed in the 1990s. While traditional restriction enzyme cloning uses short sticky ...nicks easily repairable by the bacteria after transformation. LIC is a reliable cloning method, but it is... is limited by its sequence constraints. The long overhangs must not contain the dNTP present in the exonuclease...ligation-independent cloning (SLIC), eliminated many of LIC’s constraints by relying on the power of homologous recombination...

...seven-(pass)-transmembrane domain receptors, due to the seven alpha helices that transverse the cellular...induce or inhibit intracellular signaling cascades as a response to the extracellular stimuli. Ligand dissociation...fusion to a receptor-transcription factor fusion. TEV protease cleave the transcription factor from the receptor...initiating signaling pathways in inflammation, and neurotransmission. These receptors can be activated by an array...of different ligands, including hormones, neurotransmitters, photons or odorants, to elicit downstream...coupled with heterotrimeric G proteins on the intracellular side of the cell membrane. Heterotrimeric G ...proteins are important molecular switches in signal transduction pathways and consist of a trimer of three subunits...

... by AAV? A: One potential strategy is to use split-inteins to mediate trans-protein splicing. Note, however...dose. The color diversity in Vector Assisted Spectral Tracing (VAST) experiments using PHP.eB in the brain...been able to successfully systemically transduce and then retransduce a model organism such as mice, e.g....problem? A: Re-administration is a challenge for AAV due to the development of neutralizing antibodies in... at a high MOI, often resulting in a drop in transduction coverage compared to just one vector.   Q2: ...between ddPCR and qPCR? A: At CLOVER, we have traditionally used qPCR (protocol link). We have found this... Q4: Are there any AAVs that have successful transduction in microglia? I have been using the AAV.PHP....

...to a great image is contrast. The human visual system relies primarily on contrast to identify individual...around us. Without contrast, objects simply vanish into noise. Due to its unrivaled contrast, fluorescence...fluorophores can add a second layer of contrast, color contrast, to an image and thereby provide molecular...deeply penetrating infra-red excitation light can push imaging depths closer to 1 mm. Traditionally, 2-photon... is not possible via traditional confocal due to limitations in light penetration mentioned above (5)....imaging has emerged as the dominant light microscopy contrast technique in modern biology (1). When performed...correctly, fluorescence microscopy provides a high contrast image in which a bright signal overlays a perfectly...

...and GFP reporters, as well as multiple transient and transgenic systems, Sahoo et al. showed that gene...commonly used 35S promoter. Transgenes are also efficiently inherited in both transgenic tobacco and Arabidopsis...tumor-inducing” or Ti plasmid, which allows it to transfer genetic material into the host plant genome. Scientists...Scientists have worked to optimize this system for gene transfer, studying the stability of modified Ti plasmids...a new and versatile binary Ti vector for both transient and stable gene expression applications in plants...borders specify the region of DNA that will be transferred to the plant, similar to viral vectors’ terminal...binary vectors are quite large (>10 kb.) Since transformation efficiency is inversely proportional to size...

...changing its double-strand cut to a single-stranded nick. In order to produce a double stranded break, two sgRNAs...place on the free strand of DNA and converts a C to U without producing a double strand break.   Prime... target DNA. The reverse transcriptase polymerizes DNA onto the target strand based on the sequence of... using dCas9 in bacteria demonstrated that targeting dCas9 to transcriptional start sites was sufficient...approach is that two sgRNAs targeting opposite strands must be available in moderate proximity to each...desired edit adjoined to the sgRNA, a reverse transcriptase, and a nCas9. While this system has been shown...been exploited to localize factors such as transcriptional activators and repressors to target genes. ...