The objective of this study was to examine whether the ICSI-mediated gene transfer method using in vitro matured oocytes and frozen sperm head could actually produce transgenic pigs. We also aimed at examining whether transgenic pigs can be cloned from somatic cells of a transgenic pig generated by the ICSI-mediated method. A bicistronic gene constituted of the human albumin (hALB) and enhanced green fluorescent protein (EGFP) genes was introduced into pig oocytes by the ICSI-mediated method. Transfer of 702 embryos produced by the ICSI-mediated method into five gilts resulted in 4 pregnancies. When three of the recipients, which had received total 312 of the embryos were autopsied, 32 including 1 transgenic fetuses were obtained. One of the recipients gave birth to three live piglets including one transgenic pig, showing a strong green fluorescence in the eyeballs, oral mucous membrane and subcutaneous tissues. Fluorescent microscopy revealed uniform GFP expression in all cell lines established from kidney, lung and muscle of the founder transgenic pig obtained. Nuclear transfer of these cells resulted in stable in vitro development of cloned embryos into the blastocyst stage, ranging from 12.9 to 19.8%. When 767 of the nuclear transfer embryos were transferred to 5 recipients, all became pregnant and gave birth to a total of six live transgenic-clones. The transgene copy number and integrity in the founder pig were maintained in the primary culture cells established from the founder as well as in the clones produced from these cells. Our study demonstrates that the ICSI-mediated gene transfer is an efficient and practical method to produce transgenic pigs, using frozen sperm heads and in vitro matured oocytes. It was also shown that combination of ICSI-mediated transgenesis and nuclear transfer is a feasible technology of great potential in transgenic pig production.

This study describes a cryopreservation method for porcine in vitro-produced (IVP) embryos using as a model parthenogenetic embryos derived from in vitro-matured (IVM) oocytes. IVP embryos at the expanded blastocyst stage were cryopreserved by vitrification using the minimum volume cooling (MVC) method and exhibited an embryo survival rate of 41.2%. Survival was then significantly improved (83.3%, P < 0.05) by decreasing the amount of cytoplasmic lipid droplets (delipation) prior to vitrification. IVP embryos at the 4-cell stage also survived cryopreservation when vitrified after delipation (survival rate, 36.0%), whereas post-thaw survival of nondelipated embryos was quite low (9.7%). Furthermore, it was demonstrated that porcine IVP morulae can be cryopreserved by vitrification following delipation by a noninvasive method (survival rate, 82.5%). These results clearly confirm that porcine embryos derived from IVM oocytes can be effectively cryopreserved with high embryo survival using the MVC method in conjunction with delipation.

The aim of the present study was to determine whether porcine preadipocytes can be efficient donor cells for somatic cell nuclear transfer (SCNT) in pigs. Primary culture of porcine preadipocytes was established by de-differentiating mature fat cells taken from an adult pig. The cell cycle of the preadipocytes could be synchronized by serum starvation for 1 day, with a higher efficiency than control fetal fibroblasts. Incidence of premature chromosome condensation following nuclear transfer (NT) of preadipocytes was as high as that observed after NT with fetal fibroblasts. In vitro developmental rate of the NT embryos reconstructed with preadipocyte was equivalent to that of the fetal fibroblast derived embryos. Transfer of 732 NT embryos with preadipocytes to five recipients gave rise to five cloned piglets. These data demonstrate that preadipocyites collected from an adult pig are promising nuclear donor cells for pig cloning.

To obtain a bioartificial kidney composed of a porous polymer membrane and renal cells, a polysulfone (PSf) membrane (PSM) blended with 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer was prepared. The PSM flat membrane with a porous structure could be prepared from the polymer blend containing 1 wt % of the MPC polymer in PSf by the phase inversion technique in a dry-wet process. Asymmetrical surface properties were observed on both sides of the membrane surfaces. That is, the sponge layer formed at the substrate-contacting surface of the membrane had 10-20 microm pores, but the pores in the micrometer range could not be observed for a skin layer formed at the air-contacting surface of the membrane. At the sponge layer surface, the MPC unit composition was 7 times larger than that at the skin layer surface. The amount of proteins adsorbed on the surface corresponded to the MPC unit composition. On the skin layer, a small amount of adsorbed proteins and platelet adhesion could be suppressed compared with those on the sponge layer. However, the skin layer had a moderate protein adsorption, so it showed a sufficient cytocompatibility to enable renal tubule epithelial cells to adhere and proliferate in the membrane. Thus, it functioned well as a renal tubule. Therefore, because of both its hemocompatibility and cytocompatibility, we could conclude that the PSM membrane is useful for as a renal tubule device for a bioartificial kidney.

Abstract. Incomplete cytoplasmic maturation of in vitro matured (IVM) oocytes has been known to cause microtubule and microfilament alterations, which may result in abnormal pronuclear formation and failed embryonic development. We examined the influences of maturation conditions on meiotic spindle morphology at metaphase of meiosis II (MII) in porcine oocytes. Porcine oocytes were matured under various conditions, i.e., in vitro or in vivo, with different amounts of cumulus cells, with or without hormonal supplements, and with various exposure durations to the hormones, to examine the effects on spindle morphology in MII oocytes by immunofluorescence under confocal laser microscopy. Interpolar spindle length (µm) and spindle area (µm 2 ) were compared among these maturation conditions. The spindle length was significantly shorter in IVM oocytes compared to those matured in vivo. Oocytes collected from cumulus oocyte complexes (COCs), which were poor in cumulus cells, showed smaller spindle areas than those from cumulus-rich COCs. The spindle length and area were both significantly reduced in oocytes grown without hormonal supplements. When oocytes were grown with hormonal supplements for either 6 or 22 hours for the first half of culture, there was no difference in the spindle morphology between these oocytes. These results suggested that maturation conditions significantly influence morphogenesis of MII spindles in porcine oocytes. Oocytes matured in poor conditions were more likely to have a shorter spindle length (long axis) and smaller spindle areas.