A previously functional tetracycline-regulated transactivator fails to target gene expression to the bone

Abstract

BackgroundThe tetracycline-controlled transactivator system is a powerful tool to control gene expression in vitro and to generate consistent and conditional transgenic in vivo model organisms. It has been widely used to study gene function and to explore pathological mechanisms involved in human diseases. The system permits the regulation of the expression of a target gene, both temporally and quantitatively, by the application of tetracycline or its derivative, doxycycline. In addition, it offers the possibility to restrict gene expression in a spatial fashion by utilizing tissue-specific promoters to drive the transactivator.FindingsIn this study, we report our problems using a reverse tetracycline-regulated transactivator (rtTA) in a transgenic mouse model system for the bone-specific expression of the Hutchinson-Gilford progeria syndrome mutation. Even though prior studies have been successful utilizing the same rtTA, expression analysis of the transactivator revealed insufficient activity for regulating the transgene expression in our system. The absence of transactivator could not be ascribed to differences in genetic background because mice in a mixed genetic background and in congenic mouse lines showed similar results.ConclusionsThe purpose of this study is to report our negative experience with previously functional transactivator mice, to raise caution in the use of tet-based transgenic mouse lines and to reinforce the need for controls to ensure the stable functionality of generated tetracycline-controlled transactivators over time.

Low Levels of the Reverse Transactivator Fail to Induce Target Transgene Expression in Vascular Smooth Muscle Cells

“…However, the RT-PCR amplification of human lamin A and lamin Adel150 yielded very weak signals, even after 35 cycles of amplification (Figures 1A–1C). The weak signal was also observed in samples that did not carry the reverse transactivator transgene (Figure 1B), which suggested that the weak amplification product occurred due to the leakiness of the system, which has been reported in previous studies that used the tet-ON/OFF model systems [20]–[22]. A positive control reaction was performed using cDNA synthesized from RNA extracted from the bone of a previously reported animal model containing the tet-OFF system with the same lamin A target gene, available in the lab [18].…”

“…Several investigators have reported problems using the tet-ON/OFF system and low expression of the transactivator [20] – [22] , [24] . Unfortunately, we were unable to induce transgene expression of lamin A and progerin in the different regions of aorta using the sm22α-rtTA transactivator.…”

“…Currently, we can only speculate on the reasons for the lack of transgene expression. One possibility, is that epigenetic modifications of the promotor sequence of the sm22α-rtTA transgene may have arisen over multiple mouse generations, resulting in suppression of the rtTA expression [22] , [25] – [26] .…”

Expression of the Hutchinson-Gilford Progeria Mutation during Osteoblast Development Results in Loss of Osteocytes, Irregular Mineralization, and Poor Biomechanical Properties

“…Primary osteoblasts were extracted according to the previously published procedure (34) and were processed for the in vitro mineralization assay and cytospin staining.…”

Partial Rescue of Ocular Pigment Cells and Structure by Inducible Ectopic Expression of Mitf-M in MITF-Deficient Mice

“…Although our Dct-rtTA TRE-H2BGFP system successfully results in expression of GFP in melanocytes, various causes, including integration site position of the transgene, strain background, dose responses, and epigenetic silencing, have been suggested for variegated or insufficient transgene expression in mice. 18,21–24 Although DMV mice have near normal MITF expression in the eyes in adult mice, we unfortunately cannot assess expression in cutaneous melanocytes because failure of expression (for any reason) necessarily results in an absence of skin melanocytes. Previous work has shown that expression of inducible GFP with this Dct-rtTA is higher when both transgenes are homozygous as opposed to heterozygous.…”