290 Reproduction, Fertility and Development Transgenesis Transgenesis 340 PRODUCTION OF TRANSGENIC PORCINE BLASTOCYSTS BY HANDMADE CLONING P.M. Kragh A , G. Vajta A , T.J. Corydon B , L. Bolund B , and H. Callesen A A Reproductive Biology, Department of Animal Breeding and Genetics, Danish Institute of Agricultural Sciences, 8830 Tjele, Denmark; B Institute of Human Genetics, University of Aarhus, 8000 Aarhus C, Denmark. email: peterm.kragh@agrsci.dk The present study demonstrates the application of the recently developed handmade cloning (HMC) technique in production of transgenic porcine blastocysts.The HMC technique was originally established for bovine nuclear transfer (Vajta et al., 2003, Biol. Reprod. 68, 571–578), and has the advantages of being less demanding and more productive than traditional nuclear transfer techniques. Cumulus-oocyte complexes were aspirated from slaughterhouse ovaries and matured for 41 h. Subsequently, the cumulus cells were removed by pipetting in 1 mg mL −1 hyaluronidase in HEPES-buffered TCM-199; zonae pellucidae were removed by incubation in 2 mg mL −1 pronase in HEPES-buffered TCM-199 supplemented with 2% cattle serum (T2) for 1 min. Bisection was performed by hand under a stereomicroscope using a microblade in 5 μg mL −1 cytochalasin B in TCM-199 supplemented with 20% cattle serum (T20). Demi-oocytes were incubated in 5 μg mL −1 Hoechst 33342 in T20 for 10 min, followed by examination under UV light to select the halves containing no chromatin, i.e., the cytoplasts. Porcine fibroblasts harvested from an ear skin biopsy were transfected with pN1-EGFP (Clontech) using Lipofectamine (Gibco, Life Technologies). G418 selection (0.8 mg mL −1 ) was applied 48 h after transfection, and well separated G418-resistant cell colonies originating from a single transfected cell were isolated, expanded, and cryopreserved. Days before, nuclear transfer cells were grown to a confluent monolayer in DMEM supplemented with 10% FCS. Fusions were performed 43h after start of maturation. One cytoplast was attached to one fibroblast in 500 μg mL −1 phytohemagglutinin dissolved in T2. In the fusion chamber, covered with fusion medium (0.3 M mannitol, 0.1 mM MgSO 4 , 0.05 mM CaCl 2 , and 0.01% PVA), one cytoplast-fibroblast pair was fused with one cytoplast in a single step. The fusions were performed with a double DC pulse of 65V, each pulse for 20 μs and 0.1 s apart from each other. Successfully fused embryos were activated 1 h after the end of fusion by incubation in 2 μM calcium ionophore A23187 in T20 for 5 min followed by 3-h incubation in microdrops of culture medium (NCSU-23 with 4 mg mL BSA) containing 2 mM 6-dimethylaminopurine. Activated embryos were cultured individually in microdrops of culture medium for 7 days. In four independent experiments, 93% of attempted reconstructed embryos fused and survived activation (31/31, 15/23, 28/28, and 37/37, respectively). On Day 7 after activation, the blastocyst rates (per successfully reconstructed embryos) were 6% (2/31), and 7% (1/15), 7% (2/28), and 3% (1/37), respectively. Green Fluorescent Protein was expressed in all cells of the developing blastocysts. The results show that transgenic porcine blastocysts can be produced using HMC, and the technique may also be applied for the production of transgenic pigs. 341 APOPTOSIS IN SOMATIC CELL CLONEDAND EGFPTRANSGENIC BOVINE EMBRYOS S.L. Lee, S.Y. Choe, and G.J. Rho College ofVeterinary Medicine, Gyeongsang National University, Chinju, Republic of Korea 660-701. email: jinrho@nongae.gsnu.ac.kr The overall success rate achieved by cloning techniques to date is low, mainly due to deficiencies in nuclear reprogramming, gene expression and DNA fragmentation, which result in early and late embryonic losses. This study was carried out to compare the incidences of DNA fragmentation during development of IVF, parthenote (PT), nuclear transfer (NT) and transgenic-cloned (TG) embryos.Terminal deoxynucleotidyl transferase (TdT) nick-end labelling (TUNEL) with propidium iodide counterstaining was used for determination of DNA fragmentation and total cell number. Donor cells were fetal fibroblasts with or without transfection with EGFP, and cultured in DMEM + 15% FCS until confluent, for up to 5 days. At 19 h post maturation (hpm), enucleated oocytes were reconstructed with donor cells and activated at 24hpm with the combination of ionomycin (5 μM, 5 min) and cycloheximide (10 μg/mL, 5 h) after electric fusion by a single DC pulse (1.6 KV/cm, 60 μs) delivered by a BTX 200. Parthenotes were produced by the same activation protocol at 24 hpm. The eggs and control IVF embryos were cultured in CR1aa at 39 ◦ C in a humidified atmosphere of 5% CO 2 in air. Differences among groups were analyzed using one-wayANOVA after arc-sine transformation of proportional data. Embryos at the 8-cell stage in all treatments, IVF, PT, NT and TG, showed DNA fragmentation. The apoptotic cell index (total number of apoptotic nuclei/total number of nuclei) of Day 7 blastocysts was significantly (P< 0.05) higher in TG and NT embryos (17/91, 18.6 ± 4.0% and 13/94, 13.8 ± 4.7%, respectively) compared to IVF and PT embryos (9/122, 7.4 ± 3.4% and 8/93, 8.6 ± 2.9%, respectively). TUNEL positive cells were detected in almost all blastocysts at Day 7 and were mainly observed in the ICM. The DNA fragmentation ratio of the ICM in the blastosysts at Day 7 (number of apoptotic nuclei in the ICM/total number of apoptotic nuclei in the blastocyst) was significantly (P< 0.05) higher in TG embryos (64.7 ± 21.4%) than in IVF (44.4 ± 28.0%), PT (50.0 ± 18.6%) or NT (53.0 ± 32.5%) embryos. These results indicate a higher occurrence of DNA fragmentation observed in NT and TG embryos when compared to IVF and PT embryos. In addition, ICM of TG blastocysts revealed a high DNA fragmentation ratio, which may be related to early embryonic loss after transfer of the resulting embryos. [Supported by HighTechnology Development Project forAgriculture and Forestry Korea, MAF-SGRP, 300012-05-3-SB010 and Cho-A Pharm. Co. LTD.]