...neural populations at distinct times. Temporal considerations. Temporal precision is key in optogenetic experiments... exposed to light of the correct wavelength, the pore opens, cations flow into the cell (yellow dots),...optical switches Sensors are genetically-encoded reporters of molecular signals; e.g., calcium indicators...specific subsets of neurons, allowing precise spatiotemporal control of these neurons by turning on and ...addition of trafficking signal from Kir2.1 and ER export signal provide improved membrane targeting 589 ...the vesicular gamma aminobutyric acid (GABA) transporter (VGAT) and ChR2 is thus expressed in all GABAergic...photoswitchable” proteins offer exquisite spatial and temporal control of protein activity. Phytochrome, cryptochrome...

...and a ssDNA-fluorescently quenched reporter is added. As this reporter is degraded by Cas12a, it will release... using a fluorescent reporter. In SHERLOCK, a quenched ssRNA fluorescent reporter is added to the reaction...insertions. When designing the donor template, incorporating mutations that prevent further Cas9 cleavage... HDR efficiency. In most applications, a large portion of the Cas9-induced DSBs will still be repaired...the desired HDR-edited allele. Therefore, it is important to confirm the presence of the desired edit experimentally...pairs to the 3’ flap, and the desired edit is incorporated into the DNA by reverse transcription. The edited...applications where orientation of the insert is important. Type V-K CASTs appear to exhibit almost unidirectional...

...and a ssDNA-fluorescently quenched reporter is added. As this reporter is degraded by Cas12a, it will release... using a fluorescent reporter. In SHERLOCK, a quenched ssRNA fluorescent reporter is added to the reaction...insertions. When designing the donor template, incorporating mutations that prevent further Cas9 cleavage... HDR efficiency. In most applications, a large portion of the Cas9-induced DSBs will still be repaired...the desired HDR-edited allele. Therefore, it is important to confirm the presence of the desired edit experimentally...pairs to the 3’ flap, and the desired edit is incorporated into the DNA by reverse transcription. The edited...applications where orientation of the insert is important. Type V-K CASTs appear to exhibit almost unidirectional...

...promoter is typically used for gRNA May contain reporter gene (e.g. GFP) to identify and enrich positive...vector or separate transfer vectors May contain reporter gene (e.g. GFP) or selection marker to identify... to target cells through microinjection or electroporation Transient expression of CRISPR components Expression...may alter protein function when they occur in important protein domains. For gene editing experiments ... addition to off-target activity , it is also important to consider factors that maximize cleavage of ...25408407 Hashimoto, M., & Takemoto, T. (2015). Electroporation enables the efficient mRNA delivery into the...facilitates CRISPR/Cas9-based genome editing. Scientific Reports . 5 , 11315. PMID: 26066060 Zuris, J. A., Thompson...

...promoter is typically used for gRNA May contain reporter gene (e.g. GFP) to identify and enrich positive...vector or separate transfer vectors May contain reporter gene (e.g. GFP) or selection marker to identify... to target cells through microinjection or electroporation Transient expression of CRISPR components Expression...may alter protein function when they occur in important protein domains. For gene editing experiments ... addition to off-target activity , it is also important to consider factors that maximize cleavage of ...25408407 Hashimoto, M., & Takemoto, T. (2015). Electroporation enables the efficient mRNA delivery into the...facilitates CRISPR/Cas9-based genome editing. Scientific Reports . 5 , 11315. PMID: 26066060 Zuris, J. A., Thompson...

...necessary in cases where extreme specificity is important. Their epitope specificity allows binding to a...antibodies only bind a single epitope, so it is important to choose a monoclonal antibody that will recognize...using a secondary against a tag, such as His, incorporated in the sdAb. sdAbs can have weak signals due...with conjugating reactions are possible. It is important to research your reaction and buffer before conjugating... the basic steps needed for each application, important controls, and any special considerations to think...which are bacterial proteins that bind to the Fc portion of an antibody. However, they are not suitable ... assay, selection of the fluorophores used is important. It is best to inversely pair the brightness of...

...necessary in cases where extreme specificity is important. Their epitope specificity allows binding to a...antibodies only bind a single epitope, so it is important to choose a monoclonal antibody that will recognize...using a secondary against a tag, such as His, incorporated in the sdAb. sdAbs can have weak signals due...with conjugating reactions are possible. It is important to research your reaction and buffer before conjugating... the basic steps needed for each application, important controls, and any special considerations to think...which are bacterial proteins that bind to the Fc portion of an antibody. However, they are not suitable ... assay, selection of the fluorophores used is important. It is best to inversely pair the brightness of...

...control expression of a chemogenetic reporter ensures that the reporter remains silent until a cell or tissue-specific...ligands can have high potency, which is potentially important for dosing in animal studies, made them attractive...agonists for PSAM4. A given PSAM is coupled with an ion pore domain (IPD) to form ligand-gated ion channels (...signaling. PSAM-based LGICs can be classified by the ion pore domain they are coupled to: PSAM-Gly LGICs pair ... of PSAMs and their activity in neurons PSAM Ion Pore Domain Ligand(s) Effect Outcome (in neurons) Reference...

...control expression of a chemogenetic reporter ensures that the reporter remains silent until a cell or tissue-specific...ligands can have high potency, which is potentially important for dosing in animal studies, made them attractive...agonists for PSAM4. A given PSAM is coupled with an ion pore domain (IPD) to form ligand-gated ion channels (...signaling. PSAM-based LGICs can be classified by the ion pore domain they are coupled to: PSAM-Gly LGICs pair ... of PSAMs and their activity in neurons PSAM Ion Pore Domain Ligand(s) Effect Outcome (in neurons) Reference...

... and ligated into your desired plasmid. It is important to note that restriction enzyme target sites can...be found throughout DNA sequences. Thus it is important to confirm that there are no additional sites ...complementary to the free nucleotide. Given this opportunity, T4 will resume its polymerase activity, add ...repaired by the normal replication process. It is important to note that LIC has difficulty assembling DNA...

... and ligated into your desired plasmid. It is important to note that restriction enzyme target sites can...be found throughout DNA sequences. Thus it is important to confirm that there are no additional sites ...complementary to the free nucleotide. Given this opportunity, T4 will resume its polymerase activity, add ...repaired by the normal replication process. It is important to note that LIC has difficulty assembling DNA...

...can help you begin to understand what genes are important to a certain phenotype, and allow you to design...the screening process. Amplify the library (electroporation and maxiprep). If delivering as virus, make...die or do not pass selection (in the case of a reporter) Sort “winning” cells Sequence those cells that...

...γ-retroviruses differ from lentiviruses? There are a few important differences between engineered γ-retroviruses ...small enough to enter the nucleus through nuclear pore complex. This feature may be useful for the transduction... expression of transgenes via increased nuclear export. LTR Subcomponents: U3 R U5 in cis LTR; Long terminal...

...can help you begin to understand what genes are important to a certain phenotype, and allow you to design...the screening process. Amplify the library (electroporation and maxiprep). If delivering as virus, make...die or do not pass selection (in the case of a reporter) Sort “winning” cells Sequence those cells that...

...γ-retroviruses differ from lentiviruses? There are a few important differences between engineered γ-retroviruses ...small enough to enter the nucleus through nuclear pore complex. This feature may be useful for the transduction... expression of transgenes via increased nuclear export. LTR Subcomponents: U3 R U5 in cis LTR; Long terminal...

...silencers. Promoter Regions There are three main portions that make up a promoter: core promoter, proximal...promote the initiation of transcription. The most 3' portion (closest to the gene's start codon) of the core...transcription factors bind. Distal Promoter The final portion of the promoter region is called the distal promoter...

...silencers. Promoter Regions There are three main portions that make up a promoter: core promoter, proximal...promote the initiation of transcription. The most 3' portion (closest to the gene's start codon) of the core...transcription factors bind. Distal Promoter The final portion of the promoter region is called the distal promoter...

...lentiviruses integrate into the genome. This is important because local chromatin structure at the site ...

...lentiviruses integrate into the genome. This is important because local chromatin structure at the site ...

...customized fluorescent reporters can be challenging enough, but incorporating multiple reporters into a single ... the 3’ UTR of the reporter mRNA via an MS2 coat protein (NLS-MS2-RFP) the reporter mRNA, which contains...their reporter mRNAs. Using the TRICK plasmids, Halstead et al demonstrated that 91% of reporter mRNAs... used to study transport characteristics of various Golgi and plasma membrane reporters as well as secretory... protein transport. Franck Perez’s lab has developed a whole collection of hook and reporter proteins ...pyralis) reporter plasmids for use in high-throughput screening of pre-mRNA splicing. This reporter system...widely used luciferase reporter gene plasmid pGL3 pGL3 luciferase reporter gene plasmids have been...