ReporterConstructs_F10_ - FOLTZ QUICK REF GUIDES:...

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Unformatted text preview: FOLTZ QUICK REF GUIDES: REPORTER CONSTRUCTS UPDATED 18 ­OCTOBER ­2010 1 A Primer: Basics of “Reporters” What is a reporter construct? This is piece of cDNA that encodes some (easily) detectable protein that is under the control of a promoter/enhancer of the experimenter’s choice. The experimenter delivers the DNA construct into a cell (or embryo) and then monitors the activity by looking for the presence of the reporter gene product. The detectable gene product typically encodes a fluorescent protein (like GFP, EGFP, mCherry and related) or a protein that can be easily detected with an antibody or color stain based on enzymatic activity (like LacZ, which encodes β ­galactosidase). The presence of the detectable gene product “reports” to the experimenter when and where the promoter/enhancer is expressed (if the promoter is not active, the reporter protein will not be made). See pages 40 ­41 (9th edition; pp106 ­107 8th edition) in the textbook for more info and photos of embryos expressing cell ­type specific β ­ galactosidase and GFP. What kind of promoters/enhancers are used to make reporters? There are two basic types. First is a “constitutively active” or “basal” promoter – these are used to “drive expression” of downstream coding sequence in an unregulated way. These typically can be expressed in nearly all cell types nearly all the time and at fairly high levels. These are useful when the experimenter is trying to force expression of a gene product in a cell type that normally does not express that gene (ectopic expression) or when trying to over express a given gene product ectopically. They can also be used in lineage tracing – the reporter construct can be injected into a given blastomere and then only the daughters of that cell will express the reporter (other cells are lacking the construct). Second is a “cell type specific” promoter – these are used to drive expression of the downstream reporter protein in a regulated way. Typically, there is a larger upstream regulatory region – a promoter under the control of a specific enhancer. These are “on” only in cell types that express the proper transcriptional regulators for the given enhancer, and therefore serve as excellent reporters for when and where a given promoter/enhancer is active. These are useful when the experimenter has identified a gene of interest and wants to determine when and where it is expressed – the upstream regulatory region of the gene can be used to drive expression of a reporter. These are also very useful for driving expression of gene products fused to the reporter when functional assays are called for (see below). In this case, the promoter/enhancer serves as an experimentally ­controlled device for regulating where a given gene product is produced. What is meant by a “reporter fusion”? Often, the construct will contain a promoter that drives the expression of a fusion protein – a protein of interest that is fused in ­frame with a fluorescent protein (such as GFP or mCherry). In this case, the product of the construct is a “chimeric” protein. Somewhat surprisingly, these fusion proteins are almost always functional – they work just like the normal protein even though the fluorescent protein is attached. It is critical, though, to ensure that the fusion protein FOLTZ QUICK REF GUIDES: REPORTER CONSTRUCTS UPDATED 18 ­OCTOBER ­2010 2 does function normally if the experimenter plans to use it in functional assays. This can be tested by seeing if the fusion protein construct “rescues” a knockout or knockdown of the gene of interest. What is meant by “mapping an enhancer/promoter”? Usually, genes have very large non ­coding regions. In the upstream region, the basal promoter (where the RNA polymerase machinery binds) is often identifiable based on sequence, and now we have a good idea about certain enhancer or repressor sequences as well (where transcription factors bind in order to regulate whether the RNA pol machine can bind). Enhancers can influence gene expression from thousands of base pairs away from the actual start of transcription. Often, experimenters want to identify the required sequences for proper expression. Let’s say that a given gene, X, is in hand (cloned) and that in situ hybridization analyses has shown that it is expressed (transcribed/active) only in a given lineage of cells. If we want to determine what parts of the regulatory region of the gene dictate the expression, we can construct a series of reporters that have various regions of the regulatory region driving a reporter construct. If we place these reporters in embryos and look at expression, we can determine the regulatory regions required to mimic the expression pattern we see with an in situ hyb. Finer mapping of the enhancer sequences can then be conducted using mutational analyses of the sequence to determine precisely what nucleotide sequences are required. In paper #2 (Mandel et al., 2010, Develop 137, 1919), the authors identify the regulatory element of the Xenopus Tbx20 gene by using a reporter assay (Figure 1). They then map the minimal regulatory element within this large enhancer region that recapitulates the expression of the endogenous Tbx20 gene in the heart (Figure 2). ...
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This note was uploaded on 11/11/2010 for the course MCDB 112 taught by Professor Staff during the Spring '08 term at UCSB.

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