transcriptional Protocols

Category: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

This protocol describes an in vitro transcription assay that allows for a single round of transcription from in vitro assembled chromatin. Comparing the activity of a receptor or transcriptional coactivator in an assay that measures only a single round of transcription with the results from multiple rounds of transcription can help elucidate the mechanism of transcriptional activation by those factors. - [Read Assay:Single Round of In Vitro Transcription from Assembled Chromatin Templates Using a HeLa Cell Ex]

Category: Drosophila Protocols: Drosophila Genetics Protocols

Protocol describes how to test whether a transcription factor disrupts the chromatin of a promoter of a gene of interest. First, chromatin is assembled in vitro on the gene of interest in the presence and absence of a transcriptional activator (see Protocol on Assembly of Chromatin with Drosophila S-190 Chromatin Assembly Extract and Transcriptional Activators). - [Read Nucleosomal Array Disruption Assay Protocol]

Category: Genetics and Genomics: Genotyping Protocols: PCR Genotyping

Primer pairs will amplify sequences present as a single copy in the mouse genome with the Universal Genotyping Protocol. Includes: b-Galactosidase (LacZ); cre-recombinase; CFP; diphtheria toxin; dsRED; Fabpi-200; Fabpi-500; flp recombinase; GFP/BFP/YFP; human growth hormone (complete); human growth hormone (transcriptional stop); luciferase (click-beetle); luciferase (firefly); neomycin phosphotransferase; SRY (male-specific); tTA (tet-on). - [Read PCR Genotyping Primer Pairs Protocols]

Category: Transfection Protocols: Electroporation Sonoporation Protocols

Protocol describes a method for introducing gene constructs into mouse embryos by electroporation. Gene constructs can be quickly tested for tissue-specific transcriptional activity or can be used to overexpress gene products. - [Read Protocol Electroporation]

Category: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

In this protocol nuclei isolated from cells expressing the gene of interest are incubated with radiolabeled UTP which is incorporated into nascent RNA transcripts by RNA polymerase molecules that were actively transcribing at the time the cells were harvested. Because very little denovo initiation of RNA synthesis occurs in isolated nuclei, transcription of the target gene can be measured by hybridizing the radiolabeled RNA to an excess of the target gene immobilized on a nitrocellulose or nylon - [Read Protocol for Transcriptional Run- On Assays]

Category: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

Small ubiquitin-like modifier (SUMO) is a small molecule, but has a variety of regulatory functions in cells. SUMO modification is involved in transcriptional regulation, subcellular localization, and protein-protein interactions. SUMO conjugation requires sequential E1-dependent activation, E2-dependent conjugation, and E3-dependent ligation steps. Protocol includes: In vivo and in vitro SUMOylation assay and deSUMOylation assay. - [Read Sumoylation and Desumoylation Assays for a Chromatin-Remodelling Complex In Vivo and In Vitro]

Category: Transfection Protocols: Stable Transfection Protocols

Protocol uses an autoregulatory system in which the transcriptional trans-activator tTA drives its own expression and that of a target gene. The first stage of the procedure describes how to generate stable lines of NIH-3T3 cells that express either tTA alone or tTA and the tetracycline-regulated target gene. - [Read Tetracycline as Regulator of Inducible Gene Expression Protocol]

Category: Reporter Gene Assays: Luciferase Assay Protocols

Transfection of primary leukocytes has traditionally been a challenging but much desired protocol. It allows not only the analysis of cells in a more natural state to a cell line system, it enables the direct comparison of, for e.g. transcriptional activity using luciferase reporters, in immune cells taken from genetically-altered mice. In addition, importantly it allows for "rescue experiments" in knockout cells & the ability to over-express or reconstitute wild-type and/or mutated constructs. - [Read Transfection of Bone Marrow-Derived Mast Cells for Transcription Factor Luciferase Reporter Assays]

Category: Yeast Protocols: Yeast Two-Hybrid Systems Protocols

In this stage of the protocol, a mammalian cDNA library constructed in a plasmid such as pJG4-5 is transformed into yeast strains containing pBAIT and the lexAop-lacZ reporter plasmid. PJG4-5 expresses the cloned cDNAs from a cassette containing a transcriptional activation domain and other moieties under the control of the yeast GAL1 promoter. - [Read Two-hybrid Systems Stage 2: Selecting an Interactor Protocol]