Titlebook: Gene Transfer and Expression; A Laboratory Manual Michael Kriegler Book 1990 Stockton Press 1990 Calcium.DNA.nucleic acid.protein.RNA

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Vectorsrengths and weaknesses, many of which are presented in abbreviated form in Table 2-1. In my review of these creations, I will illustrate how they can be used, as well as provide sufficient information to allow you to incorporate your own gene or cDNA inserts into these vectors. Before I describe thi

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Processing of Proteins Encoded by Transferred Genes study the gene product of interest. If that gene product is a protein molecule, you have a substantial armamentarium of methods at your disposal. In this section, I briefly describe (1) factors that can be experimentally manipulated to enhance mRNA translation, (2) the mechanism of protein processi

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DNA Transfer (2) create DNA-containing complexes whose charge characteristics are compatible with DNA uptake by the target cell; or (3) result in the transient formation of pores in the plasma membrane of a cell exposed to an electric pulse, pores of sufficient size to allow DNA to enter the target cell.

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Selection and Amplificationtion can be infected with a recombinant retrovirus. All other means of stable transformation are less efficient. To facilitate the isolation of cells stably transformed with the DNA of interest, a gene encoding a dominant selectable marker is usually included in the transfection protocol. Such a gen

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Expression Cloningis type include selectable markers and oncogenes, as well as cytokines (Wong et al., 1985) and membrane proteins including cell-surface antigens (Aruffo and Seed, 1987) and cellular receptors (D’Andrea et al., 1989).

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PCR-Based Expression chosen because it involves many of the types of problems one can run into when one chooses to pursue this approach. The solutions to these problems are also presented. In these experiments, we assembled a functional cDNA clone encoding a human leukocyte interferon a/d hybrid protein. We chose to sy

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Assays for Gene Transferferred DNA by a Southern blot of genomic DNA or, if the transferred DNA has some unique sequence feature, by the polymerase chain reaction (PCR) carried out on genomic DNA. One may also monitor successful gene transfer by assaying for mRNA production by the exogenous DNA through Northern analysis of

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