Gene Overexpression by Sense RNA in Mammalian Systems



Design and Selection of Plasmid-Based Expression Vectors Constitutive Promoter Vectors Constitutive Promoters Selectable Marker Genes Reporter Genes Splicing Regions

Kozak Sequence and Enhancer Element Inducible Promoter Vectors Retrovirus Vectors Preparation of Plasmid Sense cDNA Constructs Transient Transfection of Mammalian Cells with Sense Constructs Method A. Transfection by Calcium Phosphate Precipitation Method B. Transfection by Retrovirus Vectors

Protocol 1. Preparation of Viral Supernatant by Transient Transfection of a Packaging Cell Line with Retrovirus Vector Constructs Protocol 2. Production of Stably Transfected Producer Cell Lines

Protocol 3. Determination of Viral Titer

Protocol 4. Amplification of Virus Stock by Serial Reinfection of Fresh Target Cells

Protocol 5. Transfection of Cells of Interest with High-Titer Stock of Replication-Incompetent Retroviruses (Figure 8.9) Stable Transfection of Mammalian Cells with Sense DNA Constructs Method A. Transfection by Liposomes Method B. Transfection by Electroporation Method C. Transfection by Retrovirus Vectors Selection of Stably Transfected Cell Lines with Appropriate Drugs Use of Aminoglycoside Antibiotics Hygromycin-ß-Phosphotransferase Thymidine Kinase Tryptophan Synthetase

Characterization of Stably Transfected Cell Clones

Analysis of Gene Overexpression at the Protein Level by Western Blotting

Examination of the Expression of Sense RNA by Northern Blotting Determination of Integration Copy Number by Southern Blot Analysis

Isolation of Genomic DNA from Cultured Cells Analysis of Southern Blot Data Expression Assay of a Reporter Gene

Activity Assay of Chloramphenical Acetyl Transferase (CAT) Luciferase Assay b-Galactosidase Assay b-Galactosidase Staining of Cells Generation of Transgenic Mice from Sense ES Clones Characterization of Transgenic Mice Troubleshooting Guide References


Well-established DNA recombination and gene transfer technologies are widely used in mammalian cells and in animals to address a vast spectrum of biological questions. In order to gain an insight into the function of the gene of interest, we and others use the gene overexpression approach to up-regulate the expression of a specific gene followed by analysis of the potential roles of the gene in cell or animal systems.1-5 Gene overexpression refers to an increase in the amount of a given protein or the product level of a gene, which may result in an alteration in the function of the gene. We have successfully transfected mammalian cells with DNA constructs containing sense HSP27 cDNA and developed stably transfected cell lines in which the level of HSP27 increased four- to eightfold compared with nontransfected cells. As a result, cells containing higher levels of HSP27 are protected against heat and toxicants such as heavy metals, demonstrating that one of the functions of the HSP27 protein is to enable cells to survive and recover from stress conditions.1 Additionally, gene overexpression has a broad range of applications in animal systems.2-6 It is also applied to treat some human diseases resulting from defects in single genes by transferring the cDNA of a normal gene into patients to compensate for the lack of a given protein; this application is termed gene therapy. Therefore, gene overexpression technology constitutes one of the major advances in current medicine and molecular genetics.36-11

How is a gene of interest overexpressed? The general procedures and principles are outlined in Figure 8.1. The primary principles are that the sense cDNA constructs of interest are first introduced into cells and integrated into the chromosomal DNA of the host, and that the expression of exogenous cDNA is driven by an appropriate promoter to produce sense RNA that is translated into protein. In this way, the endogenous protein plus the exogenous protein greatly elevate the level of the protein in the cell or animal. There are two types of transfection and expression in

Construction of a cDNA library or purchase of a commercial cDNA library

Isolation and characterization of cDNA of interest by screening of the cDNA library or by PCR

Preparation of sense cDNA constructs using constitutive or inducible or retroviral vectors

Production of transgenic mice that overexpress the protein of interest

Injection of ES cells intoblastocyst-embryos

Selections and characterization of stably transfected cell lines by Southern blot, northern blot and western blot analyses

Transfection and integration of sense cDNA constructs into sense cDNA genomic DNA of host cells cDNA

Chromosomal DNA


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