Unexpected Expression Pattern of Tetracycline-Regulated Transgenes in Mice

Bao-Cutrona, Meritxell; Moral, Pedro

doi:10.1534/genetics.108.097600

Cited by 6 publications

(7 citation statements)

References 29 publications

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“…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].…”

Section: Resultssupporting

confidence: 74%

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

2014

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Hutchinson-Gilford progeria syndrome (HGPS) is a genetic disease with multiple features that are suggestive of premature aging. Most patients with HGPS carry a mutation on one of their copies of the LMNA gene. The LMNA gene encodes the lamin A and lamin C proteins, which are the major proteins of the nuclear lamina. The organs of the cardiovascular system are amongst those that are most severely affected in HGPS, undergoing a progressive depletion of vascular smooth muscle cells, and most children with HGPS die in their early teens from cardio-vascular disease and other complications from atherosclerosis. In this study, we developed a transgenic mouse model based on the tet-ON system to increase the understanding of the molecular mechanisms leading to the most lethal aspect of HGPS. To induce the expression of the most common HGPS mutation, LMNA c.1824C>T; p.G608G, in the vascular smooth muscle cells of the aortic arch and thoracic aorta, we used the previously described reverse tetracycline-controlled transactivator, sm22α-rtTA. However, the expression of the reverse sm22α-transactivator was barely detectable in the arteries, and this low level of expression was not sufficient to induce the expression of the target human lamin A minigene. The results from this study are important because they suggest caution during the use of previously functional transgenic animal models and emphasize the importance of assessing transgene expression over time.

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Section: Resultssupporting

confidence: 74%

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

2014

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“…Anti-SOD1 has been used previously (Raoul et al, 2005; Basso et al, 2006; Bao-Cutrona and Moral, 2009) and was validated and characterized in conjunction with the Human Protein Atlas (HPA) Project (HPA001401; http://www.proteinatlas.org). The next day, sections were washed and incubated in biotinylated secondary antibodies (1:1000; Vector Laboratories, Burlingame, CA).…”

Section: Methodsmentioning

confidence: 99%

Acute intermittent hypoxia induced phrenic long-term facilitation despite increased SOD1 expression in a rat model of ALS

Nichols

Satriotomo

Harrigan

et al. 2015

Experimental Neurology

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Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease characterized by motor neuron death. Since most ALS patients succumb to ventilatory failure from loss of respiratory motor neurons, any effective ALS treatment must preserve and/or restore breathing capacity. In rats over-expressing mutated superoxide dismutase-1 (SOD1G93A), the capacity to increase phrenic motor output is decreased at disease end-stage, suggesting imminent ventilatory failure. Acute intermittent hypoxia (AIH) induces phrenic long-term facilitation (pLTF), a form of spinal respiratory motor plasticity with potential to restore phrenic motor output in clinical disorders that compromise breathing. Since pLTF requires NADPH oxidase activity and reactive oxygen species (ROS) formation, it is blocked by NADPH oxidase inhibition and SOD mimetics in normal rats. Thus, we hypothesized that SOD1G93A (mutant; MT) rats do not express AIH-induced pLTF due to over-expression of active mutant superoxide dismutase-1. AIH-induced pLTF and hypoglossal (XII) LTF were assessed in young, pre-symptomatic and end-stage anesthetized MT rats and age-matched wild-type littermates. Contrary to predictions, pLTF and XII LTF were observed in MT rats at all ages; at end-stage, pLTF was actually enhanced. SOD1 levels were elevated in young and pre-symptomatic MT rats, yet superoxide accumulation in putative phrenic motor neurons (assessed with dihydroethidium) was unchanged; however, superoxide accumulation significantly decreased at end-stage. Thus, compensatory mechanisms appear to maintain ROS homoeostasis until late in disease progression, preserving AIH-induced respiratory plasticity. Following intrathecal injections of an NADPH oxidase inhibitor (apocynin; 600µM; 12µL), pLTF was abolished in pre-symptomatic, but not end-stage MT rats, demonstrating that pLTF is NADPH oxidase dependent in pre-symptomatic, but NADPH oxidase independent in end-stage MT rats. Mechanisms preserving/enhancing the capacity for pLTF in MT rats are not known.

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“…Several causes of variegated transgene expression have been proposed. Transgenes often insert as concatemers and an inverse relationship between transgene copy number and transgene silencing was reported (Bao‐Cutrona and Moral, ; Garrick et al ., ). However, repeat‐induced gene silencing was not found to be the principal reason for transgene variegation by others (Williams et al ., ) and differences in transgene copy numbers did not correlate with variegated transgene expression in the present study (Supporting Information Fig.…”

Section: Resultsmentioning

confidence: 99%

Reporter mice express green fluorescent protein at initiation of meiosis in spermatocytes

Brown

Odet

Bortner

et al. 2014

Genesis

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Transgenic mice were generated using a heat shock protein 2 (Hspa2) gene promoter to express green fluorescent protein (GFP) at the beginning of meiotic prophase I in spermatocytes. Expression was confirmed in four lines by in situ fluorescence, immunohistochemistry, western blotting, and PCR assays. The expression and distribution of the GFP and HSPA2 proteins co-localized in spermatocytes and spermatids in three lines, but GFP expression was variegated in one line (F46), being present in some clones of meiotic and post-meiotic germ cells and not in others. Fluorescence-activated cell-sorting (FACS) was used to isolate purified populations of spermatocytes and spermatids. Although bisulfite sequencing revealed differences in the DNA methylation patterns in the promoter regions of the transgene of the variegated expressing GFP line, a uniformly expressing GFP reporter line, and the Hspa2 gene, these differences did not correlate with variegated expression. The Hspa2-GFP reporter mice provide a novel tool for studies of meiosis by allowing detection of GFP in situ and in isolated spermatogenic cells. They will allow sorting of meiotic and post-meiotic germ cells for characterization of molecular features and correlation of expression of GFP with stage-specific spermatogenic cell proteins and developmental events.

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Unexpected Expression Pattern of Tetracycline-Regulated Transgenes in Mice

Cited by 6 publications

References 29 publications

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

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

Acute intermittent hypoxia induced phrenic long-term facilitation despite increased SOD1 expression in a rat model of ALS

Reporter mice express green fluorescent protein at initiation of meiosis in spermatocytes

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