...undergo retrograde transport, traveling up the axon to the cell body and driving transgene expression (Tervo...for targeting neurons, with high transduction frequencies in the central nervous system (CNS) (Wu, Asokan...  AAV8 AAV8 has high transduction efficiency in multiple tissues, rapid transgene expression, and can ... S. (2013). Analysis of transduction efficiency, tropism and axonal transport of AAV serotypes 1, 2, 5...vitreous. Science Translational Medicine, 5(189), 189ra76. https://doi.org/10.1126/scitranslmed.3005708 Goertsen...including difficult-to-target tissues like the central nervous system. In this post, we’ll dive into the... sometimes vary between target species or even strains. Pro tip! Getting a vector into a particular cell...

...cents per transformation. This protocol has worked well for us for multiple E. coli strains, including...can find the subsequent transformation protocols below: OpenWetWare Transforming chemically competent ...Untergasser Lab Transformation of chemically competent cells (protocol)  Transformation of electrocompetent... easily transformed without a heat-shock step. Because of the large number of transformations being performed...skip this incubation step and go straight to plating if your transformed cells are being plated on agar ...that transforms well in other competent cells) to ensure that your cells can be readily transformed. Compare...Want even more tips for transforming your cells? Check out our transformation guide.   ...

...most common protein-only transduction strategy is to fuse a cell penetrating peptide (CPP) to a protein...Protein-only transduction? Niels Geijsen’s group in Utrecht wanted to pursue a nonviral strategy for transducing... (78-79% after two rounds of transduction.) Compared to CCP transduction, iTOP is at least four times ..., and cell death post-transduction is low. Neuronal stem cells transduced with iTOP resulting in...lab suggests that primary cells may be better transduced using only protein. Read on for a description...description of the lab’s iTOP protein-only transduction method and its potential applications to CRISPR/Cas9 genome...the HIV protein Tat. Although CPP fusions can translocate a cell membrane, the presence of the CPP may ...

...the DNA strands. This selectively reduces the concentration of annealed mismatched substrates (expected...In this method, substrate pools were prepared consisting of one target (template) strand and four upstream...ligate many undesirable structures, including substrates containing gaps or DNA base pair mismatches (...such as Taq DNA Ligase, which only reacts with substrates containing no gaps, and will not join any fragments...generally prefer fully Watson-Crick base-paired dsDNA substrates to those containing one or more mismatches. However...pyrimidine mismatches are typically worse ligation substrates than pyrimidine:purine mismatches. Helix stability...crystal structures of several DNA ligases bound to substrate (23, 24). Thermostable DNA ligases, including ...

...gene delivery in the central nervous system due to their relatively stable extra-chromosomally long-term...anesthesia. In Cre transgenic mice, they targeted the dopamine (DA) neurons of the ventral tegmental area ...experimental constraints." Neuron 95.3 (2017): 504-529. PubMed PMID: 28772120. PubMed Central PMCID: PMC5830081...photons for energy conversion or the induction of intra- or intercellular signaling pathways (Ernst et al...the opsin into the correct orientation to be transcribed (Figure 2) (Sharma and Zhu, 2014). How does FLEx... optogenetic FLEx vectors in combination with transgenic animals or rAAVs expressing Cre under a specific...al., 2016). These very same neurons also play a central role in motivated behaviors (Juarez and Han, 2016...

...In a wet transfer system the sandwich is completely submerged in a tank filled with a transfer buffer formulated...electric current. The wet transfer process is very effective in transferring a broad range of protein ...sizes but takes hours to overnight.  In contrast, a semi-dry transfer only requires enough buffer to saturate...systems efficiently transfer a range of protein sizes in minutes without any transfer buffer. These systems...antibody binds to transiently overexpressed protein, however, they should also demonstrate that the antibody...encoding your protein of interest from Addgene, transfected it into your target cells, now what? How can ...western, proteins are: (1) separated by size, (2) transferred to a membrane, and  (3) detected using antibodies...

...PubMed PMID: 29033351. Livet, Jean, et al. "Transgenic strategies for combinatorial expression of fluorescent...labeling technique involves the introduction of a transgene construct containing fluorescent proteins (XFP...Because each cell can have multiple copies of the transgene that will recombine independently, cells may acquire...mapping of cell populations over time. The ability to track single cell dynamics at the organism level has been...non-fluorescent form of YFP as the default state of the transgene such that recombination is necessary to excise...expressed as many as 16 copies of the injected transgene, each of which could recombine to activate the...labeled, and that while multiple copies of the transgene can integrate into cells, the expression level...

... NUC HNH Cut DNA target strand RuvC Cut DNA non-target strand CTD (or PI) Recognize PAM...left) and transparent (right). The RNA guide sequence is extended as a single unpaired strand in the center...the non-target strand for the PAM sequence and doesn’t mind if the PAM complementary strand is mismatched...of the PAM in the non-target DNA strand (dark gray). Target DNA strand, light gray.   Once it has found...opaque (left) and transparent (right). Some nucleotides of the non-target DNA strand are not resolved ... sgRNA, dark blue; target DNA strand, light gray; non-target DNA strand, dark gray.   The full DNA...heteroduplex and marks the transition back to a DNA duplex. sgRNA, dark blue; target DNA strand, light gray; non-target...

...must first be transcribed into messenger RNA (mRNA). mRNA can then be translated at the ribosome into polypeptide.... Overall the general strategy for protein expression consists of transfecting cells with your DNA template...template of choice and allowing these cells to transcribe, translate and modify your protein of interest. Modified...expression post-transfection. Mammalian systems can express proteins both transiently or through stable...expression vector that is then transformed into a bacterial cell. Transformed cells propagate, are induced...including the majority of complex post-translational modifications. Extraction and purification of recombinant...vitro using purified components of the transcription and translation machinery. These include ribosomes, ...

...reagents can be delivered via transfection, nucleofection, viral transduction or injection as either protein...cell line that can be transfected at high efficiency. Lentiviral transduction Cas9 and gRNA can be present... present in a single lentiviral transfer vector or separate transfer vectors. May contain reporter gene...inserted into an AAV transfer vector and used to generate AAV particles. Transient or stable expression...Chemical transfection   Lipid mediated Cationic polymers Calcium phosphate Cells Transformed cell lines... lines Transformed cell lines Transformed cell lines Cargo Nucleic acids or protein Nucleic acids Nucleic... method: Do you need stable integration or is transient expression sufficient? Which cell types will you...

...light-dependent activity to transcription, they fused BldD to the transcriptional activators p65 and VP64 ...endogenous gene expression - demonstrating light-induced silencing of transferrin receptor CD71 in HeLa cells...readily found at Addgene. Shining light on transcriptional activation using dCas9 Initial photoactivatable...early 2015 focused on using light to control transcription. Two separate labs, Moritoshi Sato’s lab at ...photolyase homology region (CRY2PHR) fused to a transcriptional activator domain (VP64 or p65). Upon expression...the activator into position to activate gene transcription. The researchers tested a variety of combinations...LACE) system, the Gersbach lab utilized a similar strategy to develop an optimized photoactivatable CRISPR...

... differences between transformation, transduction, conjugation, and transfection Resources on Addgene.org... in 3 different ways: Transformation grade DNA Cloning grade DNA Transfection grade DNA Figure...Figure 1: Transformation introduces transformation grade plasmids into a bacterial cell. Transformation...plasmids into mammalian cells? Use transfection grade DNA. Transfection is the process of inserting an ...kits provide extra washing steps to favor the removal of this agent to increase transfection success.  In...Transformation grade DNA  Transformation is the process of inserting exogenous DNA in a microorganism. The amount ...plasmid DNA that is necessary for bacterial transformation of chemically and electrocompetent cells is...

...copy number found in plants makes traditional knockouts unattractive from a practical perspective. Therefore...non-exclusive gene silencing pathways: transcriptional and post-transcriptional gene silencing (PTGS). In either...silence the target gene(s) either transcriptionally or post-transcriptionally. In plants, different dicer-like...where approximately 20-30 nucleotides of double-stranded RNA (dsRNA) results in downregulation, or “silencing...either pathway, short double-stranded RNA fragments (siRNAs) are produced from long dsRNA via an RNase III... through targeted mRNA degradation, blocking translation, or chromatin modifications of complementary ... Figure 1: Schematic of an RNAi-inducing transgene. Typically, the desired target of RNAi is expressed...

...validated using at least one of these strategies. Choosing which strategy to use will depend on your intended... (1) other strategies may not be feasible and (2) large-scale proteomic and transcriptomic datasets already... Recombinant Strategies This approach is essentially the opposite of the Genetic strategy and looks for...Interest (GOI), and transfect it into your favorite cell line. In parallel, you transfect cells with another...(Independent) Antibodies Strategies These strategies use multiple, unique antibodies against the same ... antibody has arrived, hooray! Before you jump straight into the exciting new experiments that you’ve ...may need to roll up your sleeves and perform some extra validation experiments yourself.  Goal of Antibody...

... in vivo administration, which results in the death of transduced cells and loss of transgene expression...produced at high titers, can accommodate large transgenes, transduce quiescent and dividing cells, and do not...recombinant pAdV plasmid is identified, re-transform into a bacterial strain not prone to recombination for amplification... Keith L. March, and Bruce C. Trapnell. "Evaluation of the concentration and bioactivity of adenovirus...Adenoviral vectors (AdV) are attractive vectors for research applications and gene therapy: they can ...adenoviral genome. The genome itself is a linear double-stranded DNA of ~36kb, with inverted terminal repeats (...E3 regions and can accommodate up to 8.2 kb of transgene DNA. The second generation vectors are deficient...

...are fused to two separate proteins. (In contrast, intramolecular FRET probes contain the donor and acceptor...this FRET you speak of? Förster Resonance Energy Transfer (FRET) was originally described by Theodor Förster...alternative acronym Fluorescence Resonance Energy Transfer. Unlike the typical excitation and emission of...excited fluorophore, FRET involves a non-radiative transfer of energy (i.e. no emitted photons) from the excited...excitation by absorption of a photon 2. Energy transfer from the excited donor directly to the acceptor... acceptor In order for FRET to occur several constraints must be met. The donor and acceptor fluorophores...the acceptor fluorophore. Otherwise the energy transferred from the donor will not be able to excite the...

...the Roska lab used four different design strategies: Strategy #1 (ProA): Create promoters with sequences...specificity.  Strategy #3 (ProC): Create promoters by repeating mouse cell type specific transcription factor...were transcriptionally active in retinal cell types in mice. Figure 1: The four strategies used...synthetic promoters. Legend: TSS: transcription start site. TFBS: transcription factor binding site. Image from... sparse labeling of neurons for tracing experiments.  Neuronal tracing experiments: While synthetic promoters...for neuronal tracing experiments where lower levels of labeling makes it easier to trace a neuron’s projections...particular cell type is important not only for translational research, such as gene therapy, but also basic...

... interest.  SapTrap Tool Kit for CRISPR Cas9 edits At Addgene, we have the entire SapTrap toolkit from...17986455. PubMed Central PMCID: PMC2238901. Schwartz, Matthew L., and Erik M. Jorgensen. "SapTrap, a toolkit...26837755. PubMed Central PMCID: PMC4905529.  Schwartz, M. L., & Jorgensen, E. M. "SapTrap vectors for introducing... PubMed Central PMCID: PMC5693259.  Wang, Han, et al. "Split cGAL, an intersectional strategy using a ...PubMed Central PMCID: PMC4689222. Additional resources on the Addgene blog Learn more about SapTrap Read...scientists have been continuously sharing their worm strains through the Caenorhabditis Genetics Center (CGC...CGC) which maintains and distributes the strains all over the world. When I was at the 22nd International...

...light to regulate transcription, also includes light-regulated histone methyltransferases and deacetylases...CRISPR Epigenetics resource. Transcriptional activation p300 acetyltransferase dCas9 fused to the catalytic...other Tet1 delivery systems. Transcriptional repression DNA Methyltransferase 3 Alpha (DNMT3A) Vlatka Zoldoš...associated with transcriptional repression. Created with BioRender.com. DNA Methyltransferase MQ1 Margaret...Fusing dCas9 to a transcriptional activator like VP64 or VPR activates transcription, whereas dCas9-KRAB...domain, could mediate demethylation and induce transcription at CpG regions of various promoters. Other researchers...modifiers to specific loci without inducing double strand breaks. Below you'll find some of the CRISPR-based...

...which cleaves one strand of DNA upon binding the protospacer. The resulting double strand break (DSB) stimulates...target site, but does not cut either strand of DNA. Traditional Cas9 regulators In addition to its ...two catalytic domains, HNH and RuvC, cleave DNA strands complementary and non-complementary to the gRNA... then be fused to effector domains such as transcriptional activators or repressors. These fusion proteins...loci within a single cell. One might imagine transfecting cells with both the nuclease-positive and nuclease-null...target site (1). Although this is possible, this strategy suffers from several limitations. Perhaps most...Consequently, fewer genetic tools for targeted transcriptional and epigenetic regulation have been validated...