Stringent doxycycline dependent control of CRE recombinase in vivo

Abstract

The strategy of modulating gene activities in vivo via CRE/loxP recombination would greatly profit from subjecting the recombination event to an independent and stringent temporal control. Here, we describe a transgenic mouse line, LC-1, where the expression of the cre and luciferase gene is tightly controlled by the Tet system. Using the R26R mouse line as indicator for CRE activity, and mouse lines expressing tetracycline controlled transactivators (tTA/rtTA) in various tissues, we show that; (i) in the non-induced state CRE recombinase is tightly controlled throughout the development and adulthood of an animal; (ii) upon induction, efficient recombination occurs in the adult animal in all tissues where tTA/rtTA is present, including hepatocytes, kidney cells, neurons and T lymphocytes; and (iii) no position effect appears to be caused by the LC-1 locus. Moreover, using the novel rTA(LAP)-1 mouse line, we show that in hepatocytes, complete deletion of the loxP-flanked insert in R26R animals is achieved less than 48 h after induction. Thus, the LC-1 mouse appears suitable for exploiting two rapidly increasing collections of mouse lines of which one provides tTA/rtTA in specific cell types/tissues, and the other a variety of loxP-flanked genes.

Figure 1

Transcription units incorporated in the LC-1 and rTA^LAP-1 mouse line, respectively. (A) The bidirectional tTA/rtTA responsive promoter P_tetbi-1, present in LC-1 animals, contains an array of seven tet operator sequences flanked by two minimal promoters derived from the human cytomegalovirus promoter IE. The activated P_tetbi-1 initiates transcription of the cre and the luc gene in opposite direction. The two polyadenylation sites are derived from the human growth hormone (cre) and the SV40 early transcription unit (luc), respectively. The promoter is activated by rtTA in presence and by tTA in absence of Dox. (B) Animals of the rTA^LAP-1 mouse line contain a transcription unit consisting of the LAP promoter region which extends to –2800, a synthetic gene encoding the rtTA2^S-S2 transactivator variant, followed by the SV40 polyadenylation site. Transcriptional start sites are denoted as +1.

Figure 2

Luciferase activity in various organs of LC-1 animals in the induced and uninduced state. LC-1 animals were crossed with individuals expressing rtTA or tTA in various cell types/tissues. Luciferase was measured in extracts of tissues indicated. (A) rTA^CMV-3/LC-1, (B) TA^LAP-2/LC-1 and (C) rTA^LAP-1/LC-1 were analyzed. Induced and uninduced levels of luciferase are depicted as dark and intermediate grey columns. The light grey columns in B and C show luciferase activity in single transgenic LC-1 mice. The instrumental luciferase background around 1 r.l.u./µg of protein is indicated. The luciferase values given are the means of 4–6 animals.

Figure 3

Examination of LC-1/R26R double transgenic animals for CRE activity. Histological specimens of 14-month-old animals were stained with X-gal overnight and counterstained with nuclear fast red. No β-galactosidase activity could be detected in any organ/tissue examined of which six are exemplified here.

Figure 4

Induction of CRE recombinase in triple transgenic rTA^LAP-1/LC-1/ R26R animals. Triple transgenic 6–8-month-old mice were examined for β-galactosidase expression in the uninduced (left part, –Dox) and the induced (right part, +Dox) state. For CRE induction, animals were injected i.p. with Dox (2× 2 mg, 24 h interval). Five days after CRE induction, mice were sacrificed for histological examination. X-gal staining revealed specific patterns in liver, kidney and lung tissue which reflect the activity of the LAP promoter in the rTA^LAP-1 mouse line. In situ staining as in Figure 3.

Figure 5

PCR analysis of DNA of triple transgenic rTA^LAP-1/LC-1/R26R animals. DNA isolated from fixed tissue used for the in situ histology shown in Figure 4 was subjected to 38 amplification cycles. Only the DNA of animals where CRE was induced via Dox shows the expected recombination product of 600 bp generated by the primers R26F2 and lacZ4. lacz, control amplification product, demonstrating the presence of the lacz gene; wt, DNA from nontransgenic animals; M, molecular weight marker.

Figure 6

Identification of CRE recombinase by immunostaining. LC-1 animals crossed with either rTA^LAP-1 or TA^CamK-1 mice (line B in 24) were induced by Dox addition or withdrawal, respectively. No sign of CRE protein was detected in the uninduced state, whereas intense staining is observed upon induction in hepatocytes and neurons depending on the expression pattern of the transactivators in the respective mouse lines.