A BAC transgenic reporter recapitulates in vivo regulation of human telomerase reverse transcriptase in development and tumorigenesis

Abstract

Telomerase is tightly regulated in humans relative to mice, owing to the differential regulation of TERT genes. To explore hTERT regulation in vivo, we engineered mice with a 160-kb transgenic bacterial artificial chromosome (BAC) spanning the hTERT locus with a Renilla luciferase (Rluc) cassette downstream of its promoter. Analysis of multiple founder lines revealed that the Rluc expression profile from the transgenic hTERT reporter locus reproduced that of the native hTERT gene in all tissues and organs examined, demonstrating that genetic sequence determined the species-specific developmental regulation of the hTERT gene and that mouse epigenetic and transcription machineries faithfully regulated hTERT transcription. Thus, these mice allowed detailed analyses of developmental hTERT regulation. Both the transgenic hTERT reporter and the endogenous mTERT locus were expressed in early embryonic stages, and their mRNA levels progressively decreased throughout embryonic and postnatal development. Whereas hTERT transcription was much lower than mTERT expression in most organs, it increased significantly during postnatal development of thymus, testis, and ovary. In testis, the Rluc mRNA was enriched in elongating spermatids of seminiferous tubules. In addition, the transcription of transgenic hTERT reporter, but surprisingly not the endogenous mTERT gene, was activated during Wnt1-induced mammary tumorigenesis, allowing the monitoring of tumor development via noninvasive bioluminescent imaging. Collectively, our results demonstrate that the hTERT transgenic reporter system recapitulates the developmental regulation of the hTERT gene in a chromosomal position-independent manner and serves as a legitimate model to explore telomerase regulation in the development of normal and neoplastic tissues in vivo.

Figure 1.

Generation of transgenic hTERT reporter mice. A) Schematic illustration of the BAC reporter. BAC clone RPCI11–117B23 was used, in which an Rluc cassette was inserted at the hTERT ATG codon. Exons are designated as vertical lines. Arrows indicate transcriptional directions of the CRR9, hTERT, and Xtrp2 genes. B) Analysis of transgenic reporter locus. Top panel: diagram of the BAC reporter. Bottom panel: characterization of transgenic founders. Genomic DNAs were digested with SpeI and subjected to pulsed-field gel electrophoresis, followed by Southern blotting hybridized to probe E (47) and Rluc ORF. C) Identification of transgenic founder lines. Tail genomic DNAs were digested with PvuII and analyzed by Southern blot analysis using a mixture of Rluc and mTERT promoter probes. Numbers at bottom are copy numbers calculated based on the relative intensities of Rluc and mTERT fragments. BAC, BAC construct; M, molecular mass marker.

Figure 2.

Expression of transgenic hTERT reporters. A) Luciferase expression in adult tissues. Rluc activities in 2-mo-old A-line and D-line mice were normalized to the amounts of total proteins. B) mRNA expression in adult tissues. Total RNAs were extracted from tissues of multiple 2- to 4-mo-old D-line mice and normal human tissues. mRNA expressions were determined by qRT-PCR in triplicates, normalized to 18S rRNA. mTERT and Rluc mRNA levels in left and middle panels are expressed as percentage of those in iPSC clone III-9. Right panel shows relative levels of hTERT mRNA. *ND, not determined.

Figure 3.

In vivo expression of the transgenic hTERT reporter. A) Whole-mount in situ RNA hybridization of Rluc and mTERT mRNAs in developing embryos. E9.5 and E10.5 transgenic (D-line) and wild-type embryos were hybridized to antisense and sense riboprobes of Rluc and mTERT, as indicated. B) Whole-body BLI detection of Rluc expression in a D-line adult animal. C) Reporter expression in testis by RNA in situ hybridization. All panels except c are consecutive cryosections of a testis from a D-line male; panel c is from a nontransgenic wild-type mouse. a–e) RNA in situ hybridization using antisense (a, c, d) and sense (b, e) riboprobes of Rluc (a–c) and mTERT (d, e). g, h) Magnified images of brackets in a (g) and f (h). Dotted lines outline one seminiferous tubule. Arrowheads indicate basal layer of spermatogonial stem cells; arrows demarcate elongating spermatids. Weak Rluc signals around feet, rectal area, and oral nasal area likely resulted from autofluorescence of dried urine. Scale bars = 200 μm.

Figure 4.

Developmental regulation of the transgenic hTERT and endogenous mTERT promoters. Total RNAs were isolated from tissues and organs at different developmental stages. Levels of Rluc and mTERT mRNAs were measured by qRT-PCR, normalized to 18S rRNA. Data are shown as percentages of those in iPSC clone III-9. A) Limb and tail tips. B) Kidney, brain, lung, and heart. C) Testis, ovary, and thymus.

Figure 5.

hTERT activation in Wnt1-induced mammary tumors. A, B) Expression of transgenic hTERT reporter in mammary tumors. MTB (MMTV-rtTA)/TWNT (TetO–Wnt1)/hTERT triple-transgenic female mice A910 (60 d old) and D844 (40 d old) were put on a diet containing 2 mg/ml Dox and subjected to MNU treatment 7 d later. A910 (A) and D844 (B) mice were imaged at 36 and 46 d after MNU treatment, respectively, and tumors were dissected on the same days. Left panels: whole-body BLI of Rluc expression. Right panels: expression of Rluc and mTERT mRNAs, as well as mTERC in mammary tumors. Total RNAs were extracted from tumor biopsies and nearby normal mammary tissues. RNA levels were determined by qRT-PCR, normalized to 18S rRNA. Data are shown as fold of increase as compared to normal mammary tissue. C) RNA in situ hybridization of D2 tumor shown in B. H&E staining, riboprobes used (a), Rluc antisense (b), Rluc sense (c), mTERT antisense (d), and mTERT sense (e). Scale bars = 100 μm. D) Noninvasive imaging of mammary tumor development. MTB/TWNT/D-line triple-transgenic female mouse (81 d old) was treated with MNU after 1 wk of consuming a Dox-containing diet, followed by Dox withdrawal on d 51 postinduction. For each imaging session, the mouse was treated with 50 μg coelenteranzine in 100 μl via tail-vein injection, and Rluc images were captured, followed by IP injection of 150 μg d-luciferin and imaging of Fluc expression from the Wnt1 transgene. Triangles indicate positions of mammary tumors. Shift of the Fluc image on d 38 was from a slight movement of the mouse position during imaging. Weak Rluc signals around feet, rectal area, and oral nasal area likely resulted from autofluorescence of dried urine.