Generation of conditional mutants in higher eukaryotes by switching between the expression of two genes

Abstract

A regulatory system for the in-depth study of gene functions in higher eukaryotic cells has been developed. It is based on the tetracycline-controlled transactivators and reverse tTA, which were remodeled to discriminate efficiently between two different promoters. The system permits one to control reversibly the activity of two genes, or two alleles of a gene, in a mutually exclusive way, and also allows one to abrogate the activities of both. This dual regulatory circuit, which can be operated by a single effector substance such as doxycycline, overcomes limitations of conventional genetic approaches. The conditional mutants that can now be generated will be useful for the study of gene function in vitro and in vivo. In addition, the system may be of value for a variety of practical applications, including gene therapy.

Figure 1

Schematic outline of the Tet regulatory systems. (Upper Left) The mode of action of the tTA. tTA, a fusion protein between the Tet repressor of the Tn10 Tc resistance operon from Escherichia coli and the C-terminal portion of VP16 from Herpes simplex virus, binds in the absence of the effector molecule Dox to multiple tet operator sequences (tetO) placed upstream of a minimal human cytomegalovirus promoter and activates transcription of gene x. Addition of Dox prevents tTA from binding and thus the initiation of transcription. (Lower Left) The dose response of Dox on the tTA-dependent gene expression. Gene activity is maximal in the absence of the antibiotic, whereas increasing effector concentrations gradually decrease expression to background levels at concentrations ≥10 ng/ml. (Upper Right) The mechanism of action of the rtTA. rtTA is identical to tTA with the exception of four amino acid substitutions in the TetR moiety that convey a reverse phenotype. rtTA requires Dox for binding to tetO sequences to activate transcription of gene y. (Lower Right) The dose response of Dox on the rtTA-dependent transcription activation. There is no gene expression in the absence of the antibiotic. By increasing the effector concentration beyond 100 ng/ml Dox, rtTA-dependent gene expression is gradually stimulated.

Figure 2

Specificity of transcription activation by transactivators with new DNA-binding properties. HeLa cells kept in the absence or presence of Dox (3 μg/ml) were transiently transfected with a combination of plasmids encoding transactivators and plasmids containing the luc gene under control of tTA/rtTA responsive promoters, as indicated. The operator binding specificities of the transactivators were wt, 4C, or 6C, respectively. Correspondingly, the promoters driving the luc gene expression contained wt, 4C, or 6C tet operator sequences (P_CMV*-1, ref. , P_tet4, P_tet6). For normalization of transfection efficiencies, all DNA mixtures contained also pUHD16–1 (23) constitutively expressing the lacZ gene. After 30 h, luciferase activity in all extracts was measured and normalized to β-galactosidase activity. The figure shows transactivation of P_hCMV*-1, P_tet4, and P_tet6 by the differently specified tTAs.

Figure 3

DNA-binding and dimerization specificities of various Tc-controlled transactivators. (A) (Upper) Schematic outline of the genes encoding tTA2^B_6C and tTA2^B_4C. The 4C and 6C DNA-binding domains are located N-, the transcriptional activation domain (AD) C-terminally. When exposed to operator DNA, the two transactivators discriminate between 6C (dark), 4C (light), and 6C/4C (dark/light) operator DNA. (Lower) DNA retardation experiments (for experimental detail, see ref. 15) in which the three radio-labeled tet operator DNAs were exposed to tTA2^B_6C and tTA2^B_4C, respectively, in presence (1 μg/ml) and absence of Dox. Complex formation was detected exclusively between transactivators and their cognate operator sequences in absence of Dox. Neither tTA2^B_6C nor tTA2^B_4C binds the hybrid 6C/4C operator DNA. (B) Demonstration of heterodimer formation between tTA2^B_6C and tTA2^B_4C. (Upper) Outline of the experiment in which the two transactivators were produced simultaneously in HeLa cells. The expected heterodimers bind to the hybrid 6C/4C operator DNA, as verified in the DNA retardation experiment depicted in the lower part where 6C/4C operator DNA is shifted in absence of Dox. All designations and symbols are as in A. (C) Interaction of tTA2^E_4C with its cognate operator DNA. (Upper) Schematic outline of the experiment as in A and B; the E-class Tet repressor is indicated by stipples. The retardation experiment (Lower) shows that tTA2^E_4C discriminates well between 4C and 6C operator DNA. A faint band is, however, visible also with the composite 6C/4C operator, indicating some affinity between tTA2^E_4C and the 6C/4C operator DNA. (D) The E- and B-type transactivators do not heterodimerize (outlined, Upper). The two transactivators tTA2^E_4C and tTA2^B_6C were produced simultaneously as in B and were exposed to the various operator DNAs. Whereas both selectively bind their cognate operators, there is no sign of heterodimerization. The faint band indicating a 6C/4C operator DNA transactivator complex is caused by the low affinity of tTA2^E_4C to this DNA, as seen in C.

Figure 4

Transcription activation by tTA2^E_4C and rtTA2^B_6C at different concentrations of ATc and Tc. (A) The two plasmids encoding tTA2^E_4C and rtTA2^B_6C, respectively, were transferred into HeLa cells together with pUHC13–8 containing the luc gene controlled by P_tet4 and, for standardization of transfection efficiency, plasmid pUHD16–1 constitutively expressing lacZ (23). The transfected cultures were incubated for 36 h at the ATc concentrations indicated before cells were harvested and luciferase activity was monitored (filled circles). The result of the analogous experiment in which P_tet6 controlled the luc gene (pUHC13–9) is depicted by open circles. (B) Same experiments as described in A, except that ATc is replaced by Tc.

Figure 5

Mutually exclusive regulation of two genes by Dox. (A) Mutually exclusive expression of the luc and lacZ genes at different Dox concentrations in HT/rT-1 cells constitutively producing tTA2–1 and rtTA2–1. The plasmids carrying the luc gene under control of P_tet4 (pUHC13–8) and the lacZ gene controlled by P_tet6 (pUHG16–9) were transferred to the HT/rT-1 cells in a transient expression experiment. After incubation of the cultures for 36 h at the Dox concentrations indicated, luciferase (Top) and β-galactosidase (Middle) synthesis was monitored by indirect immunofluorescence. (Bottom) Cultures by means of phase-contrast microscopy. (B) Principle of action of the dual regulatory system. The transactivator tTA2^E_4C (simplified nomenclature: tTA2–1) activates P_tet4 in absence of Dox, whereas rtTA2^B_6C (simplified nomenclature: rtTA2–1) stimulates P_tet6 in the presence of Dox (3,000 ng/ml). At intermediate Dox levels (e. g. 30 ng/ml or at Tc concentrations between 100 and 1,000 ng/ml), none of the promoters is active. The different states of activation of the two genes x and y are indicated by small and large arrows, respectively.