Inactivating the Function of Specific Genes in Eukaryotes
■ Once a gene has been cloned, important clues about its normal function in vivo can be deduced from the observed phenotypic effects of mutating the gene.
■ Genes can be disrupted in yeast by inserting a selectable marker gene into one allele of a wild-type gene via homologous recombination, producing a heterozygous mutant. When such a heterozygote is sporulated, disruption of an essential gene will produce two nonviable haploid spores (Figure 9-37).
■ A yeast gene can be inactivated in a controlled manner by using the GAL1 promoter to shut off transcription of a gene when cells are transferred to glucose medium.
■ In mice, modified genes can be incorporated into the germ line at their original genomic location by homologous recombination, producing knockouts (see Figures 9-38 and 9-39). Mouse knockouts can provide models for human genetic diseases such as cystic fibrosis.
■ The loxP-Cre recombination system permits production of mice in which a gene is knocked out in a specific tissue.
■ In the production of transgenic cells or organisms, exogenous DNA is integrated into the host genome by non-homologous recombination (see Figure 9-41). Introduction of a dominant-negative allele in this way can functionally inactivate a gene without altering its sequence.
■ In some organisms, including the roundworm C. elegans, double-stranded RNA triggers destruction of the all the mRNA molecules with the same sequence (see Figure 9-43). This phenomenon, known as RNAi (RNA interference), provides a specific and potent means of functionally inactivating genes without altering their structure.
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