...lentiviral vector to be transported retrograde and also has higher neuronal transduction efficiency and a better...Callaway EM (2007) Monosynaptic Restriction of Transsynaptic Tracing from Single, Genetically Targeted Neurons...Lentiviral production uses three plasmids: (1) The transfer plasmid, (2) the envelope plasmid, and (3) the...the packaging plasmid. These are transfected into target cells which produce lentiviral particles.   ...preparation, VSV-G is stable and can withstand ultracentrifugation which helps scientists achieve a high titer... titer is one of the reasons VSV-G is used to transduce stem cells and neurons. However, VSV-G’s broad...for off-target infectivity as it can bind and transduce many cell types before reaching the target cells...

...can readily fold into transcriptionally active double-stranded form through intra-molecular annealing. ... Figure 2: DNA rescue and transduction of a conventional single-stranded AAV (ssAAV) and a self-complementary...genome conversion in the transduced cell nucleus, the single-to-double stranded conversion of the DNA goes...inter-molecular annealing or second strand synthesis. In contrast, a scAAV vector with half the size of...Adeno-associated virus (AAV) is a single stranded, Parvoviridae DNA virus, packaged in a non-enveloped...and in vivo laboratory experiments ranging from transient gene expression in a particular cell type to CRISPR...hijack host cell machinery, and synthesize a second strand of DNA before expression of their cargo can begin...

...22567149. Pubmed Central PMCID: PMC3342243. 4. Mathur, Ambika, et al. "Transforming training to reflect the...workforce." Science Translational Medicine (2015): 285ED4. Pubmed PMID: 25925677. Pubmed Central PMCID: PMC4484856...information on the symposium as a whole and for registration please go to http://futureofresearch.org/boston.... The Future of Research Symposium A year ago, trainees from around Boston met to discuss their visions...workforce is pyramidal/feudal generating too many trainees per PI” “Lack of awareness about how the system..., less than 15% of postdocs had achieved tenure-track positions within 5-7 years of earning their PhDs...first round of Broadening Experience in Scientific Training (BEST) awards, which are designed to support new...

...DA and other neurotransmitters in the brain has been in vivo microdialysis of extracellular fluid (Darvesh...PMID: 4403305. PubMed Central PMCID: PMC426888. Patriarchi, Tommaso, et al. "Ultrafast neuronal imaging of...dopamine (DA), a well-known reward chemical and neurotransmitter. But aside from DA’s famous role as the “happy... across the semipermeable membrane from the extracellular fluid into the perfused liquid. The probe then...is not suited to detect rapid changes in DA concentration. To address this, researchers from some labs...responses (increases in fluorescence) at low concentrations (e.g., 100 nm) of DA. In addition, they exhibit...following types of data: Report time-course and concentration of released DA in mouse brain slices Visualization...

...lateral”) gene transfer. Horizontal gene transfer can occur naturally through transformation (cellular uptake...? Plasmids 101: Transformation, Transduction, Bacterial Conjugation, and Transfection Resources on Addgene.org...environment), transduction (viral infection by bacteriophages), or conjugation (direct transfer from one cell...also promote gene transfer in soil bacteria! (Demanèche et al., 2001) Beneficial traits of natural plasmids...passengers. Most natural plasmids encode beneficial traits to entice their hosts to keep them around and even...one popular method scientists use to promote transformation, but it isn’t just for lab specimens — there...humans only noticed last century! Nevertheless, the transfer of plasmids carrying these genes between bacteria...

...PubMed Central PMCID: PMC314833. Datta, Naomi. “Transmissible Drug Resistance in an Epidemic Strain of Salmonella...factors that could be transferred between cells. At the time, these extranuclear agents of heredity were...and the labels applied to them were vague or contradictory, owing in part to the fact that very little ...Latin for 'it'), as “a generic term for any extrachromsomal hereditary determinant”. His proposal, however...1960s when scientists began to study other extrachromosomal particles, particularly Resistance or R-factors...R-factors. Like F-factors, R-factors could be transferred between bacteria via cell-to-cell contact; however...experiment. Dr. Cohen and colleagues treated a tetracycline resistant plasmid, pSC101, and a newly developed...

...only have to worry about cloning your transfer plasmid. The transfer plasmids have different packaging capacities... any HEK-293T cells. If you titrate your viral preps, lentiviral titration requires an additional couple...of days, as it requires transduction of more cells, while AAV can be titrated following purification using...the virus, or go straight into your viral application, like protein production. Titration of baculovirus...Purification Ultracentrifugation (AAV); centrifugation (lenti) Centrifugation Titration time ~1 day ...AAV and lentivirus requires three plasmids — a transfer plasmid containing your gene of interest plus ...in mammalian cells is relatively quick. After transfection, HEK-293T cells start producing virus within...

...AdV-mediated transduction of gRNA:Cas9 ribonucleoprotein complexes into transformed and non-transformed cells...(right) are transduced to target cells. Within target cells, Cas9 machinery is transiently expressed and... viruses. Adenoviruses are non-enveloped double-strand DNA viruses with a genome of around 36 kb in size...Beatty et al., 2012). These features make AdV an attractive candidate for in vivo delivery of advanced genome...left), a fragment is deleted from two homologous strands of DNA through error-prone non-homologous end joining...other viral vectors, AdVs can provide a large transgene packaging capacity. For instance, with a packaging...2014, Wang et al., 2015). In these studies, the strategy employed was to deliver a standard AdV vector ...

...discovered in positive single-stranded RNA viruses to allow for the efficient translation of multiple genes from...from a single transcript. The two strategies most widely incorporated into plasmids for research purposes...Elements Translation in eukaryotes usually begins at the 5’ cap so that only a single translation event ...scientists use a multitude of techniques including co-transfection of two or more plasmids, the use of multiple...Unlike promoters which will create unique mRNA transcripts for each gene that is expressed, multicistronic...are studying a novel gene, is not always a straightforward process. Rather than try to directly detect...your gene as they are both derived from the same transcript. Of course multicistronic vectors do not have...

...Transcription factors (TFs) act as the gate keepers for gene expression, turning transcription on and... regulatory network. The transcription atlas and library The transcription factor library The human...lentiviral particles and used to transduce human embryonic stem cells. Transduced stem cells were differentiated...approximately 1,800 TFs, with >3,500 unique isoforms transcribed from these sites. The Zhang lab generated a comprehensive...unique TF open reading frame (ORF), barcoded for tractability. These ORFs are individually overexpressed and... contribute to differentiation, the Zhang lab transduced human embryonic stem cells with the viral TF ...differentiation outcomes as well as global transcriptional changes. Using this road map, and what was ...

...dCas9 has focused on transcriptional activation using a fusion to a transcriptional activator such as VP64...repression of transcription through binding a promoter region to inhibit association of transcriptional activators... 29898790. PubMed Central PMCID: PMC6001023. Fujita, Toshitsugu, et al. "Transgenic mouse lines expressing... or TAL proteins, can be used. The Fujii lab demonstrated this in mammalian cells by expressing dCas9 ...locus of interest, the promoter region of the transcription factor IRF-1. Immunoprecipitation, using an ...2018). Furthermore, the Fujii Lab generated a transgenic mouse line and retroviral expression system for...Tapscott Lab identified regulators of DUX4, a transcriptional regulator that when inappropriately expressed...

....1260088 Damstra HG, Mohar B, Eddison M, et al (2022) Visualizing cellular and tissue ultrastructure using...publication.         Why Expansion Microscopy?  Traditional light microscopes are limited in the level of...jam packed with everything needed for synaptic transmission, are right around 250 nm, making it difficult...researcher to do if they want to understand the ultrastructural relationships that influence everything from...What is Expansion Microscopy?  ExM provides a straightforward answer to the space problem. Instead of imaging...check out a few different protocols to look for strategies that may be better for your specific targets....super-resolution” technique that uses common reagents, straightforward protocols, and standard microscopes sounds ...

... milk going to help me extract DNA from agarose gel?!”   The DNA gel extraction with glass milk method...re-used, but trace contamination could interfere with interpreting the results of transfection assays. Go... a similar workflow as a column-based plasmid extraction kit. First, you lyse the bacteria and denature...contamination and you can use a phenol chloroform extraction to remove remaining proteins and RNase. Finally...ready for use! Plasmid purification and DNA gel extraction with glass syringe filters Starting material:...fragments The plasmid purification and DNA gel extraction method described in Kim and Morrison is nearly...silica or glass, but the addition of a high concentration of a chaotropic salt (guanidine hydrochloride...

...than depletion. Multiple strategies have been developed to recruit transcriptional machinery to dCas9 and...recruit additional transcription factors. The Root and Doench labs utilized tracrRNAs with two PP7 stem...additional transcription factors to the targeted region (Sanson et al., 2018) . This tracrRNA was combined... PubMed Central PMCID: MC4253859.  4. Konermann, Silvana, et al. "Genome-scale transcriptional activation...Cas9 derivatives. Unmodified Cas9 causes double stranded DNA breaks that commonly result in a complete ... domain, such as KRAB, to prevent efficient transcription from sgRNA targets. CRISPRi is effective in ...in a narrow range around the transcription start site (TSS). The Root and Doench lab selected sgRNAs in...

...synaptic transmission. Cleavage of VAMP2 renders it inactive, thus inhibiting neurotransmitter release....depositors demonstrated the utility of light-mediated suppression of synaptic transmission in brain slices... proteins scatter sound waves and create ultrasound contrast. At a very low volumetric density and with...vivo using ultrasound in Escherichia coli and Salmonella Typhimurium. This work demonstrates engineered...Plasmids podcast Light-mediated block of synaptic transmission Article contributed by Klaus Wanisch  Listen...enable VAMP2 cleavage, and suppresses neuronal transmission within minutes. Constructs using the photodimerizer...other vital systems in the body. When a microbe travels through a host organism, the expression of some...

...mcherry) are used in high concentrations in combination with low concentrations of vectors expressing the...recombinase. The high concentration of FLEx vectors guarantees that most cells are transfected with all labeling...7624356. PubMed Central PMCID: PMC41457. 8. Schnütgen, Frank et al. 2003. “A Directional Strategy for Monitoring... sites can lead to excision, inversion, and translocation of DNA in a similar fashion as between loxP ...-LDB domains of the fusions allowing Cre/FLP translocate to the nucleus where they exert their recombinase...brain? Cells of a specific brain area can be transfected by in utero electroporation. To achieve sparse... could reduce the plasmids amounts to reduce transfection efficiency. However, if the cellular structure...

...to the strategy adopted, and to the specific reaction you are running. Transformation Transform a few ... them an E. coli  strain known to be sensitive to that antibiotic, and if the strain is growing fine, ... positive clone, try transforming the ligation mix in different bacterial strains (stbl2, NEB stable, ...The PCR could fail to produce a product, the transformation may not result in any cells, or all the colonies... will definitely help in understanding if your strategy will be successful, and to avoid easy mistakes...combination of different ways according to your cloning strategy: Restriction enzyme digestion Be mindful of the...faster cloning. Remember to quantify the DNA concentration of all the samples by gel or by nanodrop to ...

..., the ability to detect mRNA transcripts of toxins and other transcripts of interest is a benefit of this...Microbiome studies have traditionally fallen into studies of who’s there and what are they doing. To ...the organisms present while sequencing of all transcripts gives clues into what the microbes are doing.... scientists introduce an Escherichia coli donor strain into the microbiome to deliver a genetic payload... microbial species. These also contain the RP4 transfer origin as well as a selectable marker and a desired...followed by a reporter gene. The hairpin blocks translation by sequestering the RBS, but binding of the “...microbial community) reveals the RBS and allows translation of the reporter gene resulting in GFP signal....

...PubMed PMID: 25307932. PubMed Central PMCID: PMC4253859. 4. Hart, Traver, et al. "High-Resolution CRISPR...CombiGEM uses iterative one-pot reactions with straightforward restriction digestion and ligation steps to... in each plasmid. Each gRNA combination can be tracked and quantitatively analyzed by sequencing its set...expression of other genetic elements including transcription factors (Cheng et al., 2014) and microRNAs (...including those found at Addgene! Figure 1: Strategy for assembling the barcoded gRNA library pool....before beginning your experiments. Figure 2: Strategy for assembling barcoded combinatorial gRNA libraries...2014): 12462-12467. PubMed PMID: 25114216. PubMed Central PMCID: PMC4151723. 2. Doench, John G., et al. "...

...allowing the four Cre proteins to form a tetramer. A double-strand DNA break occurs within the core spacer...maintained).  Translocation: If the sites are on separate DNA molecules, a translocation event is generated... expression – placing a series of transcriptional and translational termination features with loxP sites...2839833. PubMed Central PMCID: PMC281709. 3. Tissue– and site–specific recombination in transgenic mice. Orban... pun) specific, targeted DNA modifications in transgenic animals, embryonic stem cells, and/or tissue-...recombinase”), is a 38 kDa protein responsible for intra- and inter-molecular recombination at the loxP recognition...spacer of each loxP site and the two strands are ligated, resulting in the reciprocal crossover event. This...