61 EFFICIENCY OF TWO ENUCLEATION METHODS FOR THE PRODUCTION OF TRANSGENIC PIG EMBRYOS BY HANDMADE CLONING
J. Li A B , Y.-H. Zhang A , Y.-T. Du A B , P. M. Kragh A B , S. Purup A , A. M. Pederson A , K. Villemoes A , A. L. Jørgensen B , L. Bolund B C , Q.-Z. Xue D , H.-M. Yang C and G. Vajta AA Danish Institute of Agricultural Sciences, Tjele, DK8830, Denmark
B Aarhus University, Aarhus, DK8000, Denmark
C Beijing Genomics Institute, Beijing 101300, China
D Zhejiang University, Hangzhou 310029, China
Reproduction, Fertility and Development 19(1) 148-148 https://doi.org/10.1071/RDv19n1Ab61
Submitted: 12 October 2006 Accepted: 12 October 2006 Published: 12 December 2006
Abstract
Since the successful production of transgenic pigs by somatic nuclear transfer (Lai et al. 2002 Science 295, 1089–1092), more efficient reproduction technologies for transgenic pigs have been in demand. The purpose of our work was to develop an efficient method for production of transgenic embryos by handmade cloning (HMC; Vajta et al. 2001 Cloning 3, 89–95) connected to oriented enucleation to eliminate potential harm of staining and UV illumination at cytoplast selection. After 41–42 h of in vitro maturation, oocytes were further cultured with or without 0.4 µg mL−1 demecolcine for 45 min (i.e. chemically assisted handmade enucleation (CAHE) vs. oriented handmade enucleation (OHE)). Subsequently, the cumulus cells were removed and zonae pellucidae were partially digested. Oocytes with visible extrusion cones or polar bodies attached to the surface were subjected to oriented bisection. The putative cytoplasts without extrusion cones or polar bodies, containing the major part of cytoplasm, were selected as the recipients. Two cytoplasts were electro-fused with one transgenic fibroblast expressing either amyloid precursor protein (APP) or green fluorescent protein (GFP), while non-transgenic fibroblasts were used as control nuclear donors. After activation (Kragh et al. 2005 Theriogenology 64, 1536–1545; Du et al. 2005 Cloning Stem Cells 7, 199–205), reconstructed embryos were cultured in porcine zygote medium-3 for 7 days. The rates of cleavage and blastocyst cell numbers were recorded on Day 2 and Day 7, respectively. In 5 replicates, the correct bisection efficiency achieved with CAHE was higher compared to that with the OHE method (93 ± 1% vs. 82 ± 2%, respectively; P < 0.05). Table 1 shows that blastocyst rates with APP and GFP transgenic fibroblasts as nuclear donors after CAHE were lower (P < 0.05) compared to those with the OHE method; in contrast, cleavage rates of embryos from different fibroblast donors were similar and so were blastocyst rates of non-transgenic donors after either CAHE or OHE. Our results show that embryos reconstructed from APP and GFP transgenic donors have compromised in vitro developmental rates after CAHE rather than after the OHE method; however, a high efficiency with both enucleation methods was observed when using non-transgenic somatic cells.
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