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Mosaic Gene Expression in Nuclear Transfer-Derived Embryos and the Production of Cloned Transgenic Pigs from Ear-Derived Fibroblasts¹

^a Department of Animal Sciences, University of Missouri-Columbia, Columbia, Missouri 65211 ^b Department of Veterinary Medicine, College of Animal Resource Science, Kangwon National University, Chunchon 200-701, Korea ^c State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China ^d Department of Veterinary Pathobiology, University of Missouri-Columbia, Columbia, Missouri 65211

Genetically modified domestic animals have many potential applications ranging from basic research to production agriculture. One of the goals in transgenic animal production schemes is to reliably predict the expression pattern of the foreign gene. Establishing a method to screen genetically modified embryos for transgene expression before transfer to surrogates may improve the likelihood of producing offspring with the desired expression pattern. In order to determine how transgene expression may be regulated in the early embryo, we generated porcine embryos from two distinct genetically modified cell lines by using the nuclear transfer (NT) technique. Both cell lines expressed the enhanced green fluorescent protein (eGFP); the first was a fibroblast cell line derived from the skin of a newborn pig that expressed eGFP, whereas the second was a fetal derived fibroblast cell line into which the eGFP gene was introduced by a retroviral vector. The reconstructed embryos were activated by electrical pulses and cultured in NCSU23. Although the in vitro developmental ability of each group of NT embryos was not different, the eGFP expression pattern was different. All embryos produced from the transduced fetal cell line fluoresced, but only 26% of the embryos generated from the newborn cell line fluoresced, and among those that did express eGFP, more than half had a mosaic expression pattern. This was unexpected because the fetal cell line was not clonally selected, and each cell had potentially different sites of integration. Embryos generated from the newborn cell line were surgically transferred to five surrogate gilts. One gilt delivered four female piglets, all of which expressed eGFP, and all had microsatellites identical to the donor. Here we demonstrate that transgene expression in all the blastomeres of an NT embryo is not uniform. In addition, transgene expression in a genetically manipulated embryo may not be an accurate indicator of expression in the resulting offspring.

First decision: 13 November 2001.

¹ We acknowledge funding from the F.B. Miller Fund (Department of Animal Sciences, University of Missouri-Columbia) to H.T.C., from the National Institutes of Health (NIH) via R01 RR13428 to R.S.P. and B.N.D., and from Food for the 21st Century. D.B.C. is the recipient of a Pathobiology Fellowship via T32 RR07004 funding from NIH. This work is contribution 13 184 in the Missouri Agricultural Experiment Station journal series.

² Correspondence: Randall Prather, University of Missouri, 162 ASRC, 920 East Campus Drive, Columbia, MO 65211. FAX: 573 884 7827; pratherr{at}missouri.edu

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