Key Concepts of Section 6.5

Key Concepts of Section 6.5

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 copy of a wild-type gene via homologous recombination, producing a heterozygous mutant. When such a heterozygote forms spores, two nonviable haploid spores will be produced (see Figure 6-36).

  • 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 6-38 and 6-39). Knockout mice 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 nonhomologous recombination (see Figure 6-40). Introduction of a dominant-negative allele in this way can functionally inactivate a gene without altering its sequence.

  • In many organisms, including the roundworm C. elegans, double-stranded RNA triggers destruction of the all the mRNA molecules with the same sequence (see Figure 6-42). This phenomenon, known as RNAi (RNA interference), provides a specific and potent means of functionally inactivating genes without altering their structure.

  • A bacterial system that evolved to precisely target and cleave foreign DNA, known as CRISPR, has been adapted for use in many organisms to enable specific changes to be introduced into genomic DNA. Cleavage of chromosomal DNA at a specific site by CRISPR-Cas9 usually results in a short deletion at the cleavage site. If an appropriately designed DNA segment is provided by transfection, specific DNA changes such as point mutations can be introduced at the cleavage site (see Figure 6-43).