Method for rapidly developing gene switches and gene circuits

摘要

Provided are: a selection method for a gene switch and a gene circuit, including using, as a selector, an expression vector containing at least a gene sequence whose expression is controlled by a transcription regulatory factor to be expressed when a genetic switch and a genetic circuit including the genetic switch operate, and a promoter sequence operably linked to the gene sequence upstream thereof; and an expression vector to be used in the selection method. This enables an effective selection method for a genetic switch and a genetic circuit, the selection method being able to be conducted within a short time period and with high selection efficiency and less leakiness.

主权项

1. A selection method for a genetic switch and a genetic circuit, the selection method comprising:
incubating cells transfected with
a genetic circuit expression vector comprising a genetic circuit having a genetic switch expression sequence, anda transcription regulatory factor gene sequence encoding a transcription regulatory factor whose expression is induced by a genetic switch encoded by the genetic switch expression sequence, anda controllable expression vector having a controlled gene sequence whose expression is controlled by the transcription regulatory factor, the incubation being performed in a presence or absence of an activating compound that activates the genetic switch, with addition of a first death-determining compound that induces cell death under expression of the controlled gene sequence, and collecting viable cells, and/or in a presence or absence of the activating compound with addition of a second death-determining compound that induces cell death under non-expression of the controlled gene sequence; and collecting viable cells that survive the incubation, wherein the controllable expression vector comprises a first promoter sequence operably linked to and upstream of the controlled gene sequence, and the controlled gene sequence encodes an alkylated DNA repair enzyme; and the genetic circuit expression vector comprises a target sequence of the genetic switch operably linked to the transcription regulatory factor gene sequence and a second promoter sequence different from the first promoter sequence, the transcription regulatory factor gene sequence encoding a transcription factor being operable on the first promoter sequence, and the second promoter sequence being operably linked to the genetic switch expression sequence, incubation being performed (1) when the transcription regulatory factor is a transcription repression factor, in the absence of the activating compound with addition of an alkylating agent, or (2) when the transcription regulatory factor is a transcription activation factor, in the presence of the activating compound with addition of the alkylating agent.