Tetracycline-inducible gene expression in nuclear transfer embryos derived from porcine fetal fibroblasts transformed with retrovirus vectors.
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.
Choi
Bok Ryul
BR
Division of Applied Life Science, College of Agriculture and Life Science, Gyeongsang National University, Jinju, Korea.
Koo
Bon Chul
BC
Ahn
Kwang Sung
KS
Kwon
Mo Sun
MS
Kim
Jin-Hoi
JH
Cho
Seong-Keun
SK
Kim
Kyoung Mi
KM
Kang
Jee Hyun
JH
Shim
Hosup
H
Lee
Hyuna
H
Uhm
Sang Jun
SJ
Lee
Hoon Taek
HT
Kim
Teoan
T
D000818:Animals; D004318:Doxycycline / Q000494:pharmacology; D004622:Embryo, Mammalian / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D005333:Fetus; D005347:Fibroblasts / Q000737:chemistry / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D053652:Nuclear Transfer Techniques; D012190:Retroviridae / Q000235:genetics*; D034421:Sus scrofa; D013752:Tetracycline / Q000494:pharmacology*; D014170:Transformation, Genetic; D019076:Transgenes / Q000235:genetics*
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D013752:Tetracycline; D004318:Doxycycline