... 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....

...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...

...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...

...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...

...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...

...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. ...

...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...

... 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...

....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 ...

...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...