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9 Gene Underexpression in Cultured Cells and Animals by Antisense DNA and RNA Strategies



Antisense Oligonucleotide Approaches

Design and Synthesis of Antisense Oligonucleotides Treatment of Cultured Cells with Antisense Oligomers and Determination of Optimum Dose of Oligomers by Western Blot Analysis Treatment of Cultured Cells Using an Optimum Dose of Oligomers Analysis of Inhibition of Gene Expression by Western Blotting Design and Selection of Plasmid-Based Expression Vectors Preparation of Plasmid Antisense cDNA Constructs Transient Transfection of Cultured Cells with Antisense Constructs Stable Transfection of Cultured Cells with Antisense DNA Constructs Method A. Transfection by Liposomes Method B. Transfection by Microinjection Method C. Transfection by Electroporation Method D. Transfection by Retrovirus Vectors Selection of Stably Transfected Cell Lines with Appropriate Drugs Characterization of Stably Transfected Cell Clones

Analysis of Gene Underexpression at the Protein Level by Western Blotting

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

Expression Assay of Reporter Genes Generation of Transgenic Mice

Method A. Production of Transgenic Mice from Stably Transfected ES Cells

Method B. Production of Transgenic Mice from Oocytes Characterization of Transgenic Mice Troubleshooting Guide References


In order to gain insight into the function of the protein product of a gene of interest, antisense DNA and RNA approaches have been widely applied in eukaryotes in which the antisense oligonucleotides or the antisense RNA transcripts expressed successfully inhibit the expression of specifically targeted sense RNA or mRNA.1-6 In recent years, the antisense approach has been used to address fundamental questions in molecular and cellular biology in animals, human diseases and plants.136-10 Gene underexpression refers to a decrease in the protein level of a gene, which in turn may alter the function of the gene.

Although the precise mechanism by which antisense DNA-RNA inhibits gene expression is not well known, it is generally assumed that antisense oligonucleotides or antisense RNA can interact with complementary sequence or sense RNA transcripts by base pairing, thus blocking the processing or translation of the sense RNA or mRNA. The duplex of antisense RNA and sense RNA may be rapidly degraded, resulting in a decrease in specific mRNA levels. There is no evidence, however, that the antisense RNA can directly affect the target gene or DNA sequence. Therefore, the primary principle of using antisense DNA and antisense RNA is to inhibit the expression of the target gene or mRNA level partially or completely.

How is a gene of interest underexpressed? In general, gene underexpression by antisense DNA or RNA approaches consists of highly involved techniques that include design of antisense oligonucleotides, isolation and characterization of a specific cDNA, gene transfer and analysis of gene expression in transfected cells or transgenic animals. The present chapter will focus on techniques using exogenous antisense oligonucleotides and antisense cDNA constructs to down-regulate the expression of a specific gene. These protocols have been successfully utilized in our laboratories.


Design and Synthesis of Antisense Oligonucleotides

Antisense oligomers can be chemically synthesized based on any known sequence of DNA or RNA of interest. If the DNA sequence is not known, a specific peptide sequence is available. Antisense oligonucleotides can be designed based on the amino acid sequence of the peptide.

1. Design appropriate antisense oligomers based on the target, known sequence of the mRNA (sense RNA) or its first-strand cDNA, which is the poly(T) or (-)strand. For example, if the target sequence is 5'-ACAUGCCCCUCAACGUUAGC-3' (mRNA) the antisense oligonucleotide should be designed as 3'-TGTACGGGGAGTTGCAATCG-5'. The oligo is complementary to the target sequence and both can form a hybrid as follows:


3'-TGTACGGGGAGTTGCAATCG-5' antisense oligomer

If, on the other hand, the amino acid sequence is known, the nucleotide sequence can be derived:

N—Pro-Ser-His-Gly-Arg-Ser-Pro—C amino acid sequence

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