340 PRODUCTION OF TRANSGENIC PORCINE BLASTOCYSTS BY HANDMADE CLONING

Abstract

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 43 h 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 MgSO₄, 0.05 mM CaCl₂, 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 65 V, 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.