Transgenic pig expressing the enhanced green fluorescent protein produced by nuclear transfer using colchicine‐treated fibroblasts as donor cells

Abstract

Fetal-derived fibroblast cells were transduced with replication defective vectors containing the enhanced green fluorescent protein (EGFP). The transgenic cells were treated with colchicine, which theoretically would synchronize the cells into G2/M stage, and then used as donor nuclei for nuclear transfer. The donor cells were transferred into the perivitalline space of enucleated in vitro matured porcine oocytes, and fused and activated with electrical pulses. A total of 8.3% and 28.6% of reconstructed oocytes showed nuclear envelope breakdown and premature chromosome condensation 0.5 and 2 hr after activation, respectively. Percentage of pronuclear formation was 62.5, 12 hr after activation. Most (91.4%) of the 1-cell embryos with pronuclei did not extrude a polar body. Most (77.2%) embryos on day 5 were diploid. Within 2 hr after fusion, strong fluorescence was detectable in most reconstructed oocytes (92.3%). The fluorescence in all NT embryos became weak 15 hr after fusion and disappeared when culture to 48 hr. But from day 3, cleaved embryos at the 2- to 4-cell stage started to express EGFP again. On day 7, 85.8% of cleaved embryos expressed EGFP. A total of 9.4% of reconstructed embryos developed to blastocyst stage and 71.5% of the blastoctysts expressed EGFP. After 200 reconstructed 1-cell stage embryos were transferred into four surrogate gilts, three recipients were found to be pregnant. One of them maintained to term and delivered a healthy transgenic piglet expressing EGFP. Our data suggest that the combination of transduction of somatic cells by a replication defective vector with the nuclear transfer of colchicine-treated donors is an alternative to produce transgenic pigs. Furthermore, the tissues expressing EGFP from descendents of this pig may be very useful in future studies using pigs that require genetically marked cells.

Genetically engineered Sertoli cells are able to survive allogeneic transplantation

“…28,29 The development of transgenic animals has been well established for the study of a variety of diseases and for the production of human proteins for therapeutic purposes. [30][31][32][33][34] SCs have the capacity to engraft in various environments and produce a variety of proteins that are immunoprotective. In addition, these cells are naturally very prolific protein producers.…”

Uses of GFP in Transgenic Vertebrates

“…Animal cloning has now been successful performed on the animal model systems of mouse (Wakayama et al, 1998) and zebrafish (Lee et al, 2002). This technique has also been used to successfully generate GFP transgenic livestock in goats (Reggio et al, 2001), pigs (Lai et al, 2002), and cows (Bordignon et al, 2003). The use of somatic nuclear transfer for generating transgenic organisms for research is especially promising in zebrafish.…”

Development of pig cloning studies: past, present and future

“…Moreover, the frequency of apoptosis is low in the cell subpopulations synchronized by cytodifferentiation induction compared with other synchronization systems, including serum starvation, in vitro culture to total confluency point, and chemical treatment of donor cells with specific or non-specific cyclin dependent kinase (CDK) inhibitors such as roscovitine, butyrolactone or olomoucine, which are G1/G0 phase inductors (Nagashima et al, 2003). In pig somatic cloning technology, a source of donor nuclei is more frequently the cells whose mitotic cycle is transiently inhibited at G2/M phase borderline (checkpoint) by artificial synchronization with chemical mediators from a group of kariokinetic spindle microtubule polimerization inhibitors (Lai et al, , 2002bMiyoshi et al, 2001). Partial biodegradation of microtubular cytoskeleton as a result of somatic cell incubation in a colchicine (Lai et al, 2002b) or nocodazole solution (Miyoshi et al, 2001) causes reversible blocking of the cell division cycle at metaphase or slowing down of cell proliferating activity.…”

“…In pig somatic cloning technology, a source of donor nuclei is more frequently the cells whose mitotic cycle is transiently inhibited at G2/M phase borderline (checkpoint) by artificial synchronization with chemical mediators from a group of kariokinetic spindle microtubule polimerization inhibitors (Lai et al, , 2002bMiyoshi et al, 2001). Partial biodegradation of microtubular cytoskeleton as a result of somatic cell incubation in a colchicine (Lai et al, 2002b) or nocodazole solution (Miyoshi et al, 2001) causes reversible blocking of the cell division cycle at metaphase or slowing down of cell proliferating activity. In turn a decrease of mitotic cell division rate and delay, and consequently greater uniformity of the cell growth cycle rate reduces the degree of asynchrony in entering the G2/M stage in the majority of cells of the in vitro cultured subpopulation (clonal line).…”

“…Development of the studies on pig somatic cloning, especially in recent years, was inspired not only by the necessity of quick improvement in the efficiency of the nuclear transfer technique in this species, but above all by the possibility of its practical application for multiplication of transgenic piglets, on the grounds of important implications for transplantation medicine, immunology and pharmacy. The recent reports of successful pig cloning with the use of nuclei of non-transfected differentiated cells as DNA donors (Betthauser et al, 2000;Onishi et al, 2000;Boquest et al, 2002;De Sousa et al, 2002;Walker et al, 2002) and production of transgenic pigs (expressing gene encoding enhanced green fluorescent protein) by nuclear transfer of in vitro transfected somatic cells (Park et al, 2001c(Park et al, , 2002Lai et al, 2002b;Hyun et al, 2003) may indicate the approaching phase of intensive tests on transplantation of genetically-transformed porcine organs into non-human primates and eventually human recipients (pig-to-human-xenotransplantation). So too does the production of cloned piglets generated from cultured skin fibroblasts derived from a H-transferase (α-1,2-fucosylosyltransferase; α-1,2-FT) transgenic boar (Bondioli et al, 2001).…”