42 LARGE-SCALE PRODUCTION OF CLONED TRANSGENIC PIGS: EFFICIENCY AND SIDE EFFECTS
Authors: Kurome M. , Kessler B. , Klymiuk N. , Wuensch A. , Zakhartchenko V. , Nagashima H. , Wolf E. 
Published: August 2011

Generation of transgenic pigs by somatic cell nuclear transfer (SCNT) has provided a new avenue for medical research. However, low efficiency of SCNT greatly hinders its practical applications. In this study we evaluated the efficiency of producing cloned transgenic pigs as well as the side effects of cloning observed in several piglets. As nuclear donors, we used 31 different cell cultures of porcine fetal fibroblasts, kidney cells, or skin fibroblasts derived from commercial breeds. Donor cells were modified by additive gene transfer or knockout with 21 different gene constructs or genes of interest. For additive gene transfer, a mixed cell population without previous expression analysis was used. For re-cloning, kidney or skin fibroblasts from cloned transgenic pigs were used. SCNT was performed using in vitro matured oocytes as previously reported (Kurome et al. 2006 Transgenic Res. 15, 229–240). Reconstructed oocytes were transferred laparoscopically to oestrus synchronized recipient gilts. After transfer of 9317 reconstructed oocytes into 112 recipients, 58% of recipients became pregnant and 42% of them gave birth to offspring. Birth was induced by injection of a PGF2α-analogon. Out of 159 cloned piglets, 65 (40%) were clinically healthy and developed further normally, whereas 35 (22%) cloned piglets were stillborn and 46 (30%) died soon after birth. The major reason of early neonatal death was severe underweight (<700 g), which has been found in 26 cases of several cloned litters. In rare cases, we also observed malformations like oversized tongue, cleft palate, or atresia ani. Thirteen piglets were lost because of other circumstances (killed by mother or infectious disease). The in vivo developmental competence of donor cells was analysed regarding the effects of genomic modification and re-cloning. Interestingly, we did not find significant differences in the pregnancy and delivery rates when using transfected and non-transfected cells. While the litter size from transfected cells was slightly smaller compared with non-transfected cells, the ratio of piglets per litter which survived the critical neonatal age was similar, namely 1.8 from transfected and 2.0 from non-transfected cells. Compared to the data from cloning, re-cloning resulted in lower pregnancy and delivery rates. Furthermore, efficiency with respect to the litter size or the number of clinically healthy piglets was significantly lower, as we obtained only 6 piglets from 4 pregnancies and 5 of them died at the neonatal age. In conclusion, cloned transgenic pigs were produced with several types of donor cells and genetic modifications. Nearly half of them showed abnormalities, which were not associated with the type of donor cells or the transgene used. Cloning efficiency was not affected by the procedure of genomic modification but was negatively influenced by re-cloning probably because of epigenetic alterations in the genome of donor cells.

Supported by the DFG (FOR535, FOR793), by the Bayerische Forschungsstiftung, and by Mukoviszidose e.V.