Unexpected expression pattern of tetracycline-regulated transgenes in mice

Abstract

In generating a conditional transgenic murine model based on a tetracycline-regulated system, we obtained unexpected patterns of expression due to the transcriptional inactivity of the tet-responder promoter. Here we show strong cell-type-restricted expression that was variegated to an extent determined by the number of responder transgene copies integrated into the host genome.

Figure 1.—

Experimental settings. (A) The tTA binds the core promoter (TRE; tetracycline-responder element) and activates bidirectional expression (P1, P2; minimal promoters of cytomegalovirus). Antibiotic administration (DOX; the tetracycline derivative doxycycline) abolishes transcriptional activation (OFF state). The cDNA coding for a mutated version of human SOD1 (hSOD1) was cloned into the pBI-EGFP vector (Clontech, Palo Alto, CA) and two linear constructs—4.4 kb (top: EGFP-TRE-hG93ASOD1cDNA; 997, 995, 497 transgenic lines) and 2.4 kb (bottom: TRE-hG93ASOD1cDNA; 450, 446, 428, 419, 413 transgenic lines)—were generated and micro-injected into BDF1 (C57BL/6xDBA/2) fertilized oocytes. The activator mouse line PrP-tTA F959 in the FVB/N strain (Tremblay et al. 1998) was crossed to each responder transgenic line to obtain double-transgenic mice. (B) Transiently transfected NS34 cells (green, EGFP; red, monoclonal anti-human SOD1 antibody; MBL International, Woburn, MA). A representative immunoblot is shown. Bar, 50 μm. R, responder plasmid alone; A+R, activator and responder plasmid; E, empty responder vector; NT, nontransfected cells; msod1, murine superoxide dismutase type I.

Figure 2.—

Restricted patterns of expression and doxycycline dependence. (A) Representative pattern of expression in tissue sections obtained by transcardial perfusion of double-transgenic mice (two to five animals/line). Main olfactory bulbs (MOB, M-1: bar, 800 μm; M-2–M-4: bar, 100 μm) and accessory olfactory bulbs (AOB, A-1–A-3: bar, 25 μm). Olfactory receptor neurons (O-1: bar, 10 μm) and olfactory epithelium (OE, O-2: bar, 500 μm). (B) Representative immunoblots [anti-EGFP monoclonal (Roche, Manheim, Germany); anti-SOD1 (Upstate, Lake Placid, NY); and monoclonal anti-β-actin (Immunological Sciences, Rome)]. (Left) Homogenates from double-transgenic mice from a representative EGFP-TRE-hG93ASOD1cDNA responder line in the BDF1 mouse strain background (tTA immunoblot in supplemental information). (Right) The same situation after changing the background of the same line to FVB/N (up to the eighth generation). (C) Northern blots (two to three double-transgenic mice from the three EGFP-TRE-hG93ASOD1cDNA lines). (Inset bottom right) Controls: 1, double-transgenic mouse; 2, nontransgenic mouse. (D) RT–PCR and representative tissue sections of doxycycline-treated mice dosed as indicated in drinking water with 5% sucrose over 1 week, compared to nontreated (No DOX) counterparts (three to six mice/group). The glomerular layer in the main olfactory bulb (M-1 and M-4: bar, 200 μm; M-2 and M-5: bar, 100 μm) and axonal projections (M-3 and M-6: bar, 50 μm) are shown. Stars indicate weak and low numbers of autofluorescent glomeruli. OE, olfactory epithelium; SC, spinal cord; B, brain; Cev, cerebellum; msod1 and msod2, murine superoxide dismutase types I and II.

Figure 3.—

Degree of copy-number-dependent expression variegation in the olfactory epithelium. (A) EGFP (green) signal in OMP-stained (red) sections of olfactory epithelium of EGFP-TRE-hG93ASOD1cDNA double-transgenic mice. Bars: 20 μm (997 and 497) and 30 μm (995). (B) Quantification of protein immunoblots from olfactory epithelium extracts (three to five mice/transgenic line), normalized to β-actin. (C) Efficiency of human SOD1 expression (fold levels). Protein levels normalized to β-actin and the number of responder transgene copies [N_responder (N_r), inset right] estimated by real-time PCR and Southern blotting (data not shown). Inset center: immunoblot showing decrease of hSOD1 production when responder copies increase only 10-fold. (D) Correlation between mean mRNA and protein levels based on Western and Northern blots (Figure 1C and data not shown).