Shotgun Antisense Mutagenesis

Abstract

Abstract The use of antisense RNA and DNA to block gene expression has proven to be a valuable technique for the examination of gene function in a number of different systems. Typically, a known CDNA fragment is directionally cloned into an expression vector so that cells transfected with this construct will synthesize antisense RNA transcripts. The antisense transcript will cause the loss or reduction of the corresponding gene product. The phenotypes of transformed cells are then assayed and possible functions of the gene are inferred. If, instead of transforming cells with one antisense construct, a population of cells is transformed with an antisense CDNA library, a complex pool of transformed cells is produced. This type of protocol (shotgun antisense) will yield many different clones; in each clone, the expression of a different gene product will he blocked by an antisense transcript. Mutants generated by shotgun antisense can be screened in the same manner as mutants generated by any other means. However, when a mutant is selected for further study, PCR can be used to immediately isolate the antisense CDNA, which can then be directly cloned and sequenced. This allows one to examine the sequence of the protein encoded by the repressed gene a few days after identifying a mutant with an interesting phenotype (Figure I). To confirm that the mutant phenotype was caused by the antisense CDNA construct, the PCR product isolated from the mutant of interest is cloned into the antisensc vector and used to transform cells. A matching phenotype indicates that the CDNA caused the phenotype. Alternatively, the CDNA can be used in a gene disruption construct, which in turn can be used to make a genomic knock-out of the corresponding gene. We have developed a shotgun antisense procedure for the simple eukaryote Dictyostelium discoideum (1).