The tetracycline repressor (TKR) binds to the tetracycline operator (TRE), allowing KRAB to suppress expression of the proximal gene.
Silence is golden. However, current techniques in forward genetics, including chemical mutagenesis and transposon insertion, alter but do not completely silence gene expression. Now Banks and Bradley report a forward genetic technique that knocks out entire genes in a recent article in Nature Methods.
Similar to human genetics, researchers doing forward genetics work from a known phenotype to discover the affected gene. In contrast, reverse genetics techniques, like gene knockouts, mutate a known gene and examine the resulting phenotype.
The authors designed an inducible system that randomly represses gene expression. Approximately one third of all zinc-finger, DNA-binding proteins contain Krüppel-associated box (KRAB) domains. When bound to DNA, KRAB domains recruit heterochromatin protein 1 and proteins that induce histone methylation and deacetylation. This complex produces a heterochromatin state that silences the expression of genes located within two to three kilobases. The authors designed a receptor for KRAB that inserted randomly into the genome at transcription start sites. Therefore, KRAB would repress the genes located proximal to the receptor's insertion site.
Many inducible gene expression and repression systems use bacterial receptors for the antibiotic tetracycline. The authors fused a KRAB domain to the tetracycline repressor DNA-binding protein and cloned the complex into a retroviral vector. They cloned the tetracycline operator, which contains 7 tandem repeats of the tetracycline responsive-promoter element, into a second vector. This second vector, the murine leukemia virus vector, integrates into host DNA near transcriptional start sites regardless of gene size. In cells expressing both the tetracycline repressor and operator, the tetracycline repressor binds to the tetracycline operator, allowing KRAB to repress gene expression. However, in the presence of the tetracycline analog doxycycline, the tetracycline repressor binds to its ligand, not the tetracycline operator, and transcription proceeds normally.
The authors induced expression of both the tetracycline repressor and operator vectors in cells with tetracycline-induced sensitivity to an anthrax-diphtheria hybrid toxin. They maintained the cells without doxycycline to knock out gene expression. Then they added anthrax-diptheria hybrid toxin and doxycycline. Cells that survived are presumed to lack genes necessary for anthrax and diptheria toxin-induced cell death.
Surviving cells lacked ANTXR, which encodes anthrax toxin receptors. Before toxin treatment, cells cultured without doxycycline showed reduced binding of a nontoxic truncation of anthrax toxin relative to cells cultured with doxycycline. Real-time PCR showed reduced expression of ANTXR in cells cultured without relative to cells cultured with doxycycline. In cells that survived anthrax-diptheria hybrid toxin treatment, the authors found the tetracycline operator inserted within two kilobases of the transcription start site of ANTXR.
Viral vectors are locally injected in vivo. Unlike chemical mutagenesis, this new forward genetics technique could therefore be applied in a regionally restricted manner and might allow researchers to do forward genetics in individual brain structures and nuclei.