Generation and characterization of a Tet-On (rtTA-M2) transgenic rat

Abstract

Background: The tetracycline-inducible gene regulation system is a powerful tool that allows temporal and dose-dependent regulation of target transgene expression in vitro and in vivo. Several tetracycline-inducible transgenic mouse models have been described with ubiquitous or tissue-specific expression of tetracycline-transactivator (tTA), reverse tetracycline-transactivator (rtTA) or Tet repressor (TetR). Here we describe a Tet-On transgenic rat that ubiquitously expresses rtTA-M2 driven by the murine ROSA 26 promoter.

Results: The homozygous rat line (ROSA-rtTA-M2) generated by lentiviral vector injection, has a single integration site and was derived from the offspring of a genetic mosaic founder with multiple transgene integrations. The rtTA-M2 transgene integrated into an intron of a putative gene on chromosome 2 and does not appear to affect the tissue-specificity or expression of that gene. Fibroblasts from the ROSA-rtTA-M2 rats were transduced with a TetO7/CMV-EGFP lentivirus and exhibited doxycycline dose-dependent expression of the EGFP reporter transgene, in vitro. In addition, doxycycline-inducible EGFP expression was observed, in vivo, when the TetO7/CMV-EGFP lentivirus was injected into testis, kidney and muscle tissues of ROSA-rtTA-M2 rats.

Conclusions: This conditional expression rat model may have application for transgenic overexpression or knockdown studies of gene function in development, disease and gene therapy.

Figure 1

pLenti-Rosa26-rtTA-M2 and pLenti-TetO₇/CMV-EGFP lentiviral vectors. Replication-deficient lentiviral vectors feature a self-inactivating 3'UTR and a CMV enhancer replaces the U3 region of the 5'UTR. WRE: woodchuck hepatitis posttranscriptional regulatory element. A. Expression of rtTA-M2 is driven by the ubiquitous Rosa-26 promoter. B. Expression of EGFP is under control of a seven repeat Tet operator and CMV minimal promoter (TetO₇/CMV). In the presence of doxycycline (yellow triangles), The rtTA-M2 protein (blue ovals) encoded by the construct in (A) binds the TetO₇ operator and transactivates EGFP expression from the construct in (B).

Figure 2

Integration site(s) and transgene expression analysis of rtTA-M2 transgenic rat founders. A. Southern blot analysis. Genomic DNA from four transgenic founder animals (Tg124, Tg129, Tg135, Tg144) was digested with EcoRI and hybridized with rtTA-M2 cDNA. B. Expression and function analysis of the rtTA-M2 transgene. Tail fibroblasts from the four rtTA-M2 transgenic founders were transduced with Lenti-TetO₇/CMV-EGFP and cultured in the presence of Dox (500 ng/ml). EGFP expression was observed by immunocytochemistry 48 hours post-transduction using an epifluorescent microscope and a FITC filter cube. EGFP expression was observed in transgenic founders, Tg129 and Tg144, indicating expression of the rtTA-M2 transgene and transactivation of the TetO₇/CMV-EGFP transgene. EGFP expression was not detectable in tail fibroblasts from transgenic founders Tg124 and Tg135. EGFP expression was not observed in fibroblasts from any of the transgenic founders if Dox was not added. Bar = 20 μm.

Figure 3

DNA and transgene expression analysis of F1 progeny from transgenic founder Tg144. A. Southern blot analysis. Samples of genomic DNA from Tg144 and its F1 progeny were digested with EcoRI and hybridized with rtTA-M2 cDNA. Founder (F0) and first generation progeny (F1) identification numbers are indicated at the top of each lane. Four different transgene integration patterns were passed to the F1 progeny of transgenic founder Tg144 and are identified by letter at the bottom of each lane (a, b, c, d). F1 progeny carried one to four integrations of the rtTA-M2 transgene. B. Fibroblasts from F1 progeny with each of the four integration patterns were transduced with Lenti-TetO₇/CMV-EGFP and cultured with or without Dox (Dox +/-). EGFP expression was evaluated by Western blot analysis 48 hours after viral transduction. The blot was stripped and reprobed with β-actin antibody as a loading control.

Figure 4

Characterizing rtTA-M2 transgene expression from homozygous ROSA26- rtTA-M2-1089/1088 transgenic rats (single integration). A homozygous rtTA-M2 transgenic rat line (ROSA26-rtTA-M2-1089/1099; renamed ROSA-rtTA-M2) was established by breeding F1 Tg1088 (male) and F1 Tg1089 (female), which each exhibited a single common integration of the ROSA26-rtTA-M2 transgene. A. Doxycycline dose response analysis. Fibroblasts from ROSA-rtTA-M2 F2 progeny were transduced with Lenti-TetO₇/CMV-EGFP and cultured in medium containing different concentrations of Dox (0 to 1000 ng/ml). Western blot analyses were used to evaluate EGFP expression. The blot was stripped and reprobed with β-actin antibody as a loading control. B-E. rtTA-M2 gene was stably transmitted. Fibroblasts from the F5 generation of homozygous ROSA-rtTA-M2 rats were transduced with Lenti-TetO₇/CMV-EGFP and cultured in medium without (B and C) or with (D and E) Dox (500 ng/ml). EGFP expression was observed by immunocytochemistry 48 hours post-transduction (Bar = 20 μm). F. Real time-PCR analysis to determine tissue distribution of rtTA-M2 expression from ROSA-rtTA-M2 rats. rtTA-M2 expression in each tissue is presented relative to the rtTA-M2 expression in the testis. The whiskers represent the standard errors. β-actin was used as an internal control for each tissue.

Figure 5

Rosa-rtTA-M2 integrated into a putative gene on chromosome 2. A. Rosa-rtTA-M2 provirus inserted into intron 11 of putative gene (GeneID: 310880) on chromosome 2. E: Exon. B. Genomic PCR using primers from Exon 10 (P1) and vector (P2) verified this integration. Lane M: 1 Kb DNA ladder; lane 2, no digestion; lane 3, EcoRI digestion; lane 4, Spe I digestion; lane 5, wide type rat DNA). C. Detection of the putative endogenous gene expression in rat tissues by RT-PCR. Reverse transcription was performed with an oligo (dT)₁₇ primer. The endogenous transcript was amplified using primers from exon 10 (P1) and exon 13 (P3) of this putative gene. M2: ROSA-rtTA-M2 rat; wt: wild type rat.

Figure 6

Evaluation of EGFP expression in tissues from ROSA-rtTA-M2 transgenic rat. Lenti-TetO₇/CMV-EGFP was injected into the testis, hind leg skeletal muscle or kidney of ROSA-rtTA-M2 rats without (A, C, E and G) or with (B, D, F and H) Dox in their drinking water (0.5 mg/ml). A-B. Immunohistochemical analysis of EGFP expression in histological section of testicular seminiferous tubule. Counterstain: DAPI. C-D. Immunohistochemical analysis of EGFP expression in whole mount of testicular seminiferous tubule. E-F. Immunohistochemical analysis of EGFP expression in skeletal muscle. G-H. Immunohistochemical analysis of EGFP expression in Kidney. Counterstain: hematoxylin. Bar = 50 μm (all panels).