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Tetracycline‐inducible gene expression in nuclear transfer embryos derived from porcine fetal fibroblasts transformed with retrovirus vectors
Author(s) -
Choi Bok Ryul,
Koo Bon Chul,
Ahn Kwang Sung,
Kwon Mo Sun,
Kim JinHoi,
Cho SeongKeun,
Kim Kyoung Mi,
Kang Jee Hyun,
Shim Hosup,
Lee Hyuna,
Uhm Sang Jun,
Lee Hoon Taek,
Kim Teoan
Publication year - 2006
Publication title -
molecular reproduction and development
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.745
H-Index - 105
eISSN - 1098-2795
pISSN - 1040-452X
DOI - 10.1002/mrd.20543
Subject(s) - biology , transgene , doxycycline , microbiology and biotechnology , gene expression , green fluorescent protein , fibroblast , somatic cell nuclear transfer , reporter gene , transgenesis , embryo , gene , regulation of gene expression , cell culture , genetics , embryogenesis , reproductive technology , blastocyst , antibiotics
A critical problem of transgenic livestock production is uncontrollable constitutive expression of the foreign gene, which usually results in serious physiological disturbances in transgenic animals. One of the best solutions for this problem may be use of controllable gene expression system. In this study, using retrovirus vectors designed to express the enhanced green fluorescent protein ( EGFP ) gene under the control of the tetracycline‐inducible promoter, we examined whether the expression of the transgene could be controllable in fibroblast cells and nuclear transfer (NT) embryos of porcine. Transformed fibroblast cells were cultured in medium supplemented with or without doxycycline (a tetracycline analog) for 48 hr, and the induction efficiency was measured by comparing EGFP gene expression using epifluorescence microscopy and Western and Northern blot analyses. After the addition of doxycycline, EGFP expression increased up to 17‐fold. The nuclei of transformed fibroblast cells were transferred into enucleated oocytes. Fluorescence emission data revealed strong EGFP gene expression in embryos cultured with doxycycline, but little or no expression in the absence of the antibiotic. Our results demonstrate the successful regulation of transgene expression in porcine nuclear transfer embryos, and support the application of an inducible expression system in transgenic pig production to solve the inherent problems of side‐effects due to constitutive expression of the transgene. Mol. Reprod. Dev. © 2006 Wiley‐Liss, Inc.