NE102 Lecture Notes 2

Gene transcribed translated into protein vector

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Gene = Transcribed & translated into protein.  Vector = Serves as a vector (or carrier) for the promoter and gene sequences.  Recombinant plasmids are often created to express a protein of interest. Called  expression plasmids OVERRIDING QUESTION: How can we use recombinant DNA technology to introduce  prtoeins into cells to study cell function? Can be introduced into cells. Two common methods
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Recombinant DNA pt. 2 19:00 Recombinant plasmids can be designed & engineered to contain specific “types” of DNA  sequences.  The particular “types” of sequence engineered into a plasmid depends on what it is to  be used for. The most common use for recombinant plasmids is to introduce a protein of interest into  cells. Called “Expression plasmids” or “Expression constructs” Three fundamental types of sequence in expression plasmids: Promoter, gene of interest, plasmid vector Promoters – activate transcription of gene of interest (From viral genes) Gene of interest – Gets transcribed into protein of interest Plasmid vector – Serves as a carrier for the promoter and gene sequences Expression plasmids can be inserted into cells to introduce your protein of interest QUESTION: How can expression plasmids be inserted into cells? Two methods: (1) Transfection: use of chemical reagent engineered to deliver DNA into cells (2) Infection: Plasmid packaged into a virus, which can deliver DNA into cells QUESTION: How would you introduce p53 protein into cells to study is function? Generate an expression plasmid that codes for p53. Add a transfection reagent or infect the cells How might you ensure that p53 is actually being introduced? How about immunoblot?
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Recombinant DNA pt. 2 19:00 Have to run a control that was not transfected with p53 to make sure that p53 was  coming from plasmid and not the cell itself. P53 is “ overexpressed ” in cells transfected with the p53 plasmid AFTER SPRING BREAK IN LAB: Examine cells transfected with an expression plasmid  encoding a mutant form of the protein  Ras Already known:  Ras  - protein that is activated when PC12 cells are treated with NGF Ras-G12V  – a mutant Ras protein that is constitutively active Not subject to normal modes of regulation QUESTION: Does Ras induce neuronal differentiation? Add a transfection reagent and you will determine if cells differentiate based on “neurite  outgrowth”. COMMON APPLICATIONS OF RECOMBINANT DNA 1. To introduce a protein of interest into cells (via expression plasmids) 2. To introduce green fluorescent protein (aka GFP) into cells.  What is GFP?  Protein discovered in jellyfish that fluoresces green  Has many uses Marker for cell transfection
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