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

Abstract

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.

Pig transgenesis by Sleeping Beauty DNA transposition

“…Retro-and lentiviral vectors are established potent tools for stable modification of porcine somatic donor cells for SCNT Park et al 2001;Park et al 2002) and have been successfully used for transgene delivery to porcine embryos (Cabot et al 2001;Hofmann et al 2003;Whitelaw et al 2004). SB-directed transgenesis combined with SCNT and HMC represents a nonviral alternative that offers insertion of a defined genetic unit, strong systemic transgene expression, and possibilities of multicopy transgene insertion.…”

“…Permanent genetic modification of somatic donor cells for SCNT has been achieved by random genomic insertion of plasmid DNA (Hyun et al 2003;Kragh et al 2009;Kragh et al 2004;Watanabe et al 2005) or by genomic integration of transduced retroviral or lentiviral vectors Park et al 2001;Park et al 2002). In both non-viral and viral approaches inclusion of a drug resistance gene in the transgene-encoding vectors allows selection for cells containing the integrated vector.…”

Cheetah interspecific SCNT followed by embryo aggregation improves in vitro development but not pluripotent gene expression

“…This phenomenon could be due to higher cell numbers from the beginning of embryo culture, or to an epigenetic combination within each embryo that compensates for inefficient cellular reprogramming of individual embryos, or to a combination of both hypotheses. The epigenetic combination obtained after the aggregation of two genetically identical reconstructed embryos that were reprogrammed differently (Boiani et al 2002, Park et al 2002, could compensate for defective individual embryos enhancing developmental competence of aggregates (Eckardt & McLaughlin 2004, Balbach et al 2012. In this manner, aggregation could make the development of a complete embryo possible, even if one of the two contributing embryos may not have been competent alone.…”

“…As a result of the donor nucleus and recipient ooplast state, each reconstructed embryo is unique in terms of epigenetic marks and gene expression (Park et al 2002). This characteristic affects embryo quality and consequently cloning success.…”

Differential reprogramming of somatic cell nuclei after transfer into mouse cleavage stage blastomeres

“…Continuation of reprogramming during cleavage stages may either be mediated by maternal factors remaining from the ooplasm or via novel gene expression from a partially reprogrammed somatic nucleus. Observations such as mosaic gene expression in murine and porcine blastocyst stage clones could indicate either differential reprogramming between blastomeres or a differential failure of gene expression (Boiani et al 2002, Park et al 2002. Differential reprogramming between blastomeres would require that modifications occur subsequent to the first cleavage.…”