神經科學概論
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(2) Why not sequence everything?. • The cloning of the gene encoding proteins that are responsible for known neuronal activities can yield important information about the structure, function and regulation of these moieties.. 2.
(3) Visualizing neuronal gene expression. • Molecular neuroscience depends on our ability to monitor and measure the expression of genes.. 3.
(4) The aim of the technique applied in molecular neuroscience • • • • •. To enable gene products to be assayed Qualitatively Quantitatively Any point of the expression pathway From transcription of the gene to the generation of the mature, functional peptide product. • What is the sensitivity of the technique? • What is the anatomical resolution of the technique? • Is the technique quantitative? • Does the method tell us anything about the structure of the gene product? • How can the results be interpreted in terms of gene functions?. 4.
(5) • How the standard methods used in most molecular neuroscience laboratories to visualize gene expression measure? • How these technologies will develop in the coming years? • How might gene expression be monitored within the brain of a living organism? • Could this ever be achieved non-invasively?. National Human Genome Research Institute (NHGRI). 5.
(6) Monitoring and measuring transcription. Methods to Measure Gene Expression Gene. Nuclear run-on assay +Southern blot. 6.
(7) Nuclear run-on assay RNA polymerase II complexes Rradioactive ribonucleotide Nascent RNA. Monitoring and measuring RNA. 7.
(8) Methods to Measure Gene Expression Gene RNA. Nuclear run-on assay +Southern blot Northern blot RT-PCR Real time RT-PCR In situ hybridization Microarray. RT-PCR. 8.
(9) Real Time RT-PCR. In Situ Hybridization. Detection of mRNA in tissue sections using labelled complementary cloned DNA. National Human Genome Research Institute (NHGRI). by artist Darryl Leja. 9.
(10) Microarray. National Human Genome Research Institute (NHGRI) by artist Darryl Leja. Monitoring and measuring protein. 10.
(11) Methods to Measure Gene Expression Gene RNA. Protein. Nuclear run-on assay +Southern blot Northern blot RT-PCR Real time RT-PCR In situ hybridization Microarray Enzyme assay Electrophoresis (SDS-PAGE) Immunoassay (ELISA) Western blot Immunocytochemistry. Enzyme Assay • Measurement of any biochemical reaction in which product appearance or substrate disappearance due to enzyme activity of interested protein can be measured. – colorimetric reactions using substrates which produce a colored product with the enzyme – radioactive substrates. 11.
(12) Immunoassay (ELISA). ELISA - (enzyme-linked immunosorbent assay) Quantification of protein amounts using antibodies conjugated to enzymes which can be assayed with a colorimetric or fluorimetric substrate and its color is proportional to amount of protein product. Electrophoresis (SDS-PAGE). 12.
(13) Electrophoresis (SDS-PAGE). Western blot 1. PAGE to separate proteins. 2. Transfer of proteins to membrane. 3. Detection of proteins by specific antibody. 13.
(14) Detection System for Western Blot. Immunocytochemistry. Detection of specific protein expression in tissue sections using labelled antibodies.. 14.
(15) Monitoring and measuring protein • The presence of an authentic RNA in a cell is not necessarily indicative of peptide synthesis or biologically active peptide – Poor translation – Posttranslational processes are absent – The peptide is rapidly degraded. Gene expression Î Protein expression Î Protein function. 15.
(16) Monitoring and measuring protein • Methods study the expression of protein in the brain – Radioimmunoassay – Western blotting – Immunocytochemistry. Radioimmunoassay (RIA) • Samples can be fractionated by column chromatography Æ RIA • The binding of a radioactively labeled antigen to a fixed amount of antibody can be inhibited by the addition of unlabeled antigen, and the extent of inhibition is a measure of the unlabeled material added. 16.
(17) Western blotting • Samples can be fractionated in SDSpolyacrylamide gel Æ electrophoretic separation Æ protein mass. 17.
(18) Immunocytochemistry • To localize the peptide product of a gene • To identify particular cell types within tissue sections • Detection – Enzyme – Fluorescent tag – Gold particle. Co-localization • Whether the particular cells within the CNS express two or more genes of interest.? • In situ hybridization • Immunocytochemistry • Combine In situ hybridization and Immunocytochemistry. 18.
(19) • To correlate the expression of gene transcripts with functional properties of specific individual neurons in the brain – Combine the patch-clamp technique and RT-PCR. Neuroscience Functions vs gene products. 19.
(20)
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