34 PIGLETS BORN FROM HANDMADE CLONING

Abstract

Somatic cell nuclear transfer (SCNT) is probably the most efficient way to produce pigs with targeted genetic modification. Handmade cloning (HMC) is a new technology for SCNT developed recently (Vajta et al. 2005 Reprod. Fertil. Dev. 17, 97–112). However, HMC that resulted in births in cattle was regarded technically difficult in pigs due predominantly to the fragility of MII phase porcine oocytes. The purpose of our present work was to use optimized porcine HMC for production of cloned piglets. Data were analyzed by t-test using SPSS (11.0; SPSS Inc., Chicago, IL, USA). After 42 h in vitro maturation, cumulus cells were removed. Oriented handmade enucleation was performed as described elsewhere (Li et al. concomitant abstract). Briefly, oocytes that were partially digested with 3.3 mg mL-1 of pronase for 20 s were removed of polar bodies (PB) and adjacent small volume of cytoplasm by manual bisection in HEPES-buffered TCM-199 medium supplemented with 2% calf serum and 2.5 �g mL-1 of cytochalasin B. Halves without PB were collected as putative cytoplasts. In vitro-cultured porcine fetal fibroblasts were used as donor cells. After cytoplast-fibroblast pairing, fusion and activation of fused cytoplast-fibroblast pairs (Kragh et al. 2005 Theriogenology 64, 1536–1545; Du et al. 2005 Clon Stem Cells 7, 199–205), reconstructed embryos were cultured in a modified well of the well (WOW) culture system (Feltrin et al. 2006 Reprod. Fertil. Dev. 18, 126 abst) with porcine zygote medium-3 (PZM3) supplemented with 4 mg mL-1 of BSA. The cumulative effect of the optimization steps has resulted in considerably improved in vitro efficiency, shown as 64 � 2.3 (mean � SEM) reconstructed embryos from 151.3 � 4.8 oocytes could be obtained per day after 3–4 h manual work, including a 1-h pause between fusion and activation. One-half (50.1 � 2.8%) of the reconstructed embryos developed to the blastocyst stage after 7 days, a rate that was significantly higher than that obtained with traditional cloning (TC; 27.7 � 2.2%; P < 0.01). To compare the transfer efficiency between HMC and TC, blastocysts from both HMC and TC produced by using nuclear donor cells of different origin, respectively, to identify the offspring were transferred surgically to synchronized recipients. Of 6 pregnancies produced, 2 are ongoing, 2 were lost, and 2 term litters of 3 and 10 piglets were born. Live birth/transferred embryo efficiencies for HMC and TC are 17% (10/58) and 15% (3/20). According to our knowledge, a litter size of 10 cloned healthy piglets, as achieved in this study, is the highest one that ever has been reported. Our data suggest that porcine HMC is a very promising method for SCNT and may promote its widespread application for various purposes.