...goal of this year’s workshop is to brainstorm practical strategies for using combinations of AAV-based...result of the meeting will be a publicly shared practical guide for all scientists in the field and every...Organizing Committee: Dr. Constance Cepko, Harvard Medical School Dr. Melina Fan, Dr. Susanna Bachle, Dr. ...Addgene Confirmed Speakers Constance Cepko Harvard Medical School Ali Cetin Allen Institute for Brain Science...technology is used for targeted neuronal manipulation. Practical tips for using the technology in the lab (e.g....publication, we are asking participants to share practical information in the form of data and protocols ...publication's permission). Connie Cepko, Harvard Medical School Ali Cetin and Shenqin Yao, Allen Institute...

...it's usually more efficient than plasmid donor. Typical design is to have 50-80bp of homology arms on each... changes (>100bp insertions or deletions), we typically use plasmid donor, with two homology arms on each...transfection in cells like HEK, we use around 400ng of a typical size (~ 5kb) donor plasmid with Cas9/sgRNAs vector...plasmids. For ssDNA oligo design, the Zhang lab typically uses around 100-150bp total homology. The mutation..., giving 50-75bp homology arms. The Zhang lab typically use PAGE purified long oligos. For large changes...100bp insertions or deletions), the Zhang lab typically uses a plasmid donor, with two homology arms on...like HEK, the Zhang lab uses around 400ng of a typical size (~5kb) donor plasmid with Cas9/sgRNAs vector...

... by Talley Lambert at Harvard Medical School Zeiss Microscopy : Optical Highlighters NIC Wiki PSFP : Nikon...Protein Resources: FRET Biosensors Subcellular localization Michael Davidson Collection Blog: Which FP Should...Monomer? Background Fluorescent proteins are genetically encoded tools that are used extensively by life... proteins created by Talley Lambert at Harvard Medical School More References below Jump to: Blue/UV Cyan... tagging with a wide variety of colors, some specifically optimized for yeast Lindquist Lab Vectors - ...Nature Methods July 2016, Vol. 13(7):557-62 Olympus Confocal : The Fluorescent Protein Color Palette NIC Wiki... Shcherbakova et al. : Journal of the American Chemical Society, May 2012, Vol. 134 No.18, pp. 7913–7923...

...gRNA Design Tools CRISPR Blog Posts Base Edit Catalytically dead dCas9 fused to a cytidine deaminase protein...Promoter Selectable Marker PI Publication Activate Catalytically dead dCas9 fused to a transcriptional activator... Selectable Marker PI Publication Interfere Catalytically dead dCas9, or dCas9 fused to a transcriptional...epigenetic modifications, researchers have fused catalytically dead dCas9 to epigenetic modifiers. Design your...domain of ADAR2(E488Q) adenosine deaminase to catalytically dead Cas13b creates a programmable RNA editor...Insert Selectable Marker PI Publication Purify A catalytically inactive Cas9 (dCas9) can be used to purify ...Selectable Marker PI Publication Visualize A catalytically inactive Cas9 (dCas9) fused to a fluorescent...

...) domains that respond specifically to small molecules termed Pharmacologically Selective Effector Molecules.... Optogenetics Chemogenetics plasmids encode genetically engineered receptors that are activated by specific... to artificially activate or inhibit specific biological processes. Addgene’s collection contains two ...Activated by Designer Drugs (DREADDs) and Pharmacologically Selective Actuator Modules (PSAMs). DREADDs...have been engineered to respond to specific physiologically inert small molecules. There are four types...

..., excitatory and inhibitory opsins , genetically-encoded calcium indicators, and rabies targeting genes...doubly-specified combination of genetic and/or anatomical-defined parameters, by placing two orthogonal...fragments (which have become exons) determines the logical expression requirements of Cre and Flp (B,E). When...Fenno LE, Ramakrishnan C, Kim YS, Deisseroth K, Callaway EM, Oberhuber M, Conzelmann KK, Staiger JF. 2019...Parvalbumin-Expressing GABAergic Neurons in Barrel Cortex Reveals Local and Long-Range Circuit Motifs. Cell Rep. 28(13):...Robertson K, Osten P, Sabatini BL. 2019. Distinct Cortical-Thalamic-Striatal Circuits through the Parafascicular...

...incorporate a non-canonical amino acid into the protein of interest: The non-canonical amino acid, which...aaRS) that has been evolved to incorporate non-canonical amino acids (ncAAs). A plasmid containing the ...the gene of interest with the modified codon (typically the amber codon) that is recognized by the cognate...plasmids have been introduced in the cells, the non-canonical amino acid can be incorporated using the existing...acid onto the tRNA. The tRNA and synthetase are called an orthogonal set, because they should not crosstalk...the tRNA with a different amino acid. They are typically expressed from a single plasmid, with multiple...to specific stimuli, such as light. On a broader scale, the expanded genetic code can help us understand...

...to help identify guide sequences that minimize identical genomic matches or near-matches to reduce risk...band” is often too large to efficiently amplify. Typically use primers at least 100 bp from the predicted... to final volume of 50 μl. This reaction may be scaled down to a smaller final volume if necessary. Run...inoculate into a maxi-prep culture. Prep size may be scaled based on the required DNA yield. Perform maxi-prep...clones have sufficiently proliferated once macroscopically visible with ~2 mm diameter. Primer Validation...cells) before screening individual clones. It is critical that the deletion and non-deletion amplicons be...Masuda T, Maeda T, Paw BH, Orkin SH. Journal of Biological Chemistry . 2014. PubMed . Correction of a genetic...

...’s disease (PD) is a chronic and progressive neurological disorder that primarily affects movement. More...-wide association studies (GWAS) or other large scale studies. Researchers are now investigating the role...indicators with improved activation kinetics and localization for imaging synaptic transmission. Aggarwal ... transcription factors for sensory, motor, and cortical neuron differentiation. Held et al. Cell Rep. ...fluorescent proteins, including Markers of Subcellular Localization, Optogenetics Plasmids, and Chemogenetics Plasmids...(Link opens in a new window) A collection of neurological mouse models including complex and monogenic...

... Megason 13769 pCALNL-DsRed Cre dependent DsRed expression Mammalian Cepko 13770 pCALNL-GFP Cre dependent...Mammalian Sato 122961 pcDNA3.1_PA-Cre-Y324F Catalytically inactive PA-Cre CMV Mammalian Sato 123128 pET-His-Cre...140135 pAAV-EF1a-iCreV Split Cre fused to nuclear localized wild-type VVD EF-1 alpha AAV Zeng 140284 pCAG-...stop codon with loxP sites on either side (often called a “lox-stop-lox” or “LSL” cassette) upstream of...putting the loxP sites on either side of a gene (called “floxing”, for “flanked by loxP”), will permit ...excise the shRNA to return gene expression to physiological levels. In lox-STOP-lox shRNA constructs, Cre...which reporters are initially OFF and then probabilistically ON or OFF following Cre recombination to maximize...