Hollow fiber vitrification: a novel method for vitrifying multiple embryos in a single device.

Current embryo vitrification methods with proven efficacy are based on the minimum volume cooling (MVC) concept by which embryos are vitrified and rewarmed ultrarapidly in a very small amount of cryopreserving solution to ensure the high viability of the embryos. However, these methods are not suitable for simultaneously vitrifying a large number of embryos. Here, we describe a novel vitrification method based on use of a hollow fiber device, which can easily hold as many as 40 mouse or 20 porcine embryos in less than 0.1 µl of solution. Survival rates of up to 100% were obtained for mouse embryos vitrified in the presence of 15% DMSO, 15% ethylene glycol and 0.5 M sucrose using the hollow fiber vitrification (HFV) method, regardless of the developmental stage of the embryos (1-cell, 2-cell, morula or blastocyst; n = 50/group). The HFV method was also proven to be effective for vitrifying porcine in vitro- and in vivo-derived embryos that are known to be highly cryosensitive. For porcine embryos, the blastocyst formation rate of in vitro maturation (IVM)-derived parthenogenetic morulae after vitrification (48/65, 73.8%) did not decrease significantly compared with non-vitrified embryos (59/65, 90.8%). Transfer of 72 in vivo-derived embryos vitrified at the morula/early blastocyst stages to 3 recipients gave rise to 29 (40.3%) piglets. These data demonstrate that the HFV method enables simultaneous vitrification of multiple embryos while still adhering to the MVC concept, and this new method is very effective for cryopreserving embryos of mice and pigs.

Knockout of exogenous EGFP gene in porcine somatic cells using zinc-finger nucleases.

Zinc-finger nucleases (ZFNs) are expected as a powerful tool for generating gene knockouts in laboratory and domestic animals. Currently, it is unclear whether this technology can be utilized for knocking-out genes in pigs. Here, we investigated whether knockout (KO) events in which ZFNs recognize and cleave a target sequence occur in porcine primary cultured somatic cells that harbor the exogenous enhanced green fluorescent protein (EGFP) gene. ZFN-encoding mRNA designed to target the EGFP gene was introduced by electroporation into the cell. Using the Surveyor nuclease assay and flow cytometric analysis, we confirmed ZFN-induced cleavage of the target sequence and the disappearance of EGFP fluorescence expression in ZFN-treated cells. In addition, sequence analysis revealed that ZFN-induced mutations such as base substitution, deletion, or insertion were generated in the ZFN cleavage site of EGFP-expression negative cells that were cloned from ZFN-treated cells, thereby showing it was possible to disrupt (i.e., knock out) the function of the EGFP gene in porcine somatic cells. To our knowledge, this study provides the first evidence that the ZFN-KO system can be applied to pigs. These findings may open a new avenue to the creation of gene KO pigs using ZFN-treated cells and somatic cell nuclear transfer.

Developmental ability of porcine in vitro matured oocytes at the meiosis II stage after vitrification.

The aim of the present study was to investigate whether a combination of cytoplasmic lipid removal (delipation) and treatment by a microtubule stabilizer, paclitaxel, would lead to efficient cryopreservation of porcine in vitro matured (IVM) oocytes at the meiosis II (MII) stage. Vitrification and subsequent re-warming and culture of 109 untreated oocytes produced only 9 blastocysts (8.3%). On the other hand, the post-vitrification blastocyst rate was significantly improved (21/113, 18.6%, P<0.05) when oocytes were treated with 1 microM paclitaxel. Oocyte delipation also significantly increased the post-vitrification blastocyst rate compared with the untreated group (15/37, 40.5%, P<0.05). The delipation-and-paclitaxel group exhibited a significantly higher blastocyst rate (34/75, 45.3%, P<0.05) than the paclitaxel group, although it was not significantly higher than that for the delipation group. In transfer experiment, a total of 109 (18.6%) parthenogenetic blastocysts were obtained from 586 oocytes vitrified with the delipation-and-paclitaxel treatment. Transfer of 72 blastocysts to two recipients resulted in 14 (19.4%) somite stage fetuses. In conclusion, we demonstrated for the first time that by removing cytoplasmic lipid droplets from oocytes and performing a microtubule stabilization procedure, vitrified porcine IVM MII-stage oocytes could efficiently develop to the blastocyst stage while retaining the ability to develop into fetuses.