Abstract
Pigs are anatomically and physiologically closer to humans than other laboratory animals. Transgenic (TG) pigs are widely used as models of human diseases. The aim of this study was to produce pigs expressing a tetracycline (Tet)-inducible transgene. The Tet-on system was first tested in infected donor cells. Porcine fetal fibroblasts were infected with a universal doxycycline-inducible vector containing the target gene enhanced green fluorescent protein (eGFP). At 1 day after treatment with 1 µg/ml doxycycline, the fluorescence intensity of these cells was increased. Somatic cell nuclear transfer (SCNT) was then performed using these donor cells. The Tet-on system was then tested in the generated porcine SCNT-TG embryos. Of 4,951 porcine SCNT-TG embryos generated, 850 were cultured in the presence of 1 µg/ml doxycycline in vitro. All of these embryos expressed eGFP and 15 embryos developed to blastocyst stage. The remaining 4,101 embryos were transferred to thirty three surrogate pigs from which thirty eight cloned TG piglets were obtained. PCR analysis showed that the transgene was inserted into the genome of each of these piglets. Two TG fibroblast cell lines were established from these TG piglets, and these cells were used as donor cells for re-cloning. The re-cloned SCNT embryos expressed the eGFP transgene under the control of doxycycline. These data show that the expression of transgenes in cloned TG pigs can be regulated by the Tet-on/off systems.
Highlights
Transgenic (TG) pigs generated using assisted reproductive techniques are a major research tool in xenotransplantation [1,2,3], disease models [4,5], and bioreactors [6,7]
When the cells were treated with 1 mg/ml doxycycline, enhanced green fluorescent protein (eGFP) fluorescence was first detected on day 2 and peaked on day 3 (Fig. 1b). eGFP expression gradually decreased when doxycycline was removed from the culture medium for up to 3 days (Fig. 1b)
PCR analyses using genomic DNA isolated from eGFP-positive FACS-sorted primary fetal fibroblasts (pFF) as the template and a pair of primers designed to amplify eGFP confirmed that eGFP was inserted into the genome of these cells
Summary
Transgenic (TG) pigs generated using assisted reproductive techniques are a major research tool in xenotransplantation [1,2,3], disease models [4,5], and bioreactors [6,7]. In some TG pigs generated using the standard procedure, organ function and embryo developments are abnormal; for example, overexpression of an exogenous GH gene causes a range of pathophysiological abnormalities [13] To overcome these problems, systems that allow inducible expression of transgenes are required. A modified reverse tetracycline (Tet)-controlled transactivator protein (rtTA) has been used to produce a wide range of TG animals including pig [17], dog [18], and chicken [19]. This system is more sensitive to doxycycline and yields lower background expression than the original rtTA system. An advanced system that uses a reverse rtTA (Tet-on) promoter has been developed that behaves in the opposite way [20]
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