23 Production of Genetically Modified Founder Pigs as Models for Human Diseases

Abstract

Pigs are increasingly used as genetically modified (GM) animal models for human diseases. Reliable methods to produce GM piglets are needed to produce at least one founder animal that can pass on the transgene to the next generation using conventional reproductive procedures. Somatic cell nuclear transfer (SCNT, “cloning”) is one such method, although it has a low efficiency with up to only 10% of offspring born based on number of cloned embryos transferred (Liu et al. 2014 Reprod. Fertil. Dev. 27, 429-439) and with a high percentage dying in the first days after birth (Schmidt et al. 2015 Theriogenology 84, 1014-1023). Furthermore, there is concern about the normality and viability of offspring in the following generations after cloning. Here, we report our results related to the latter question and describe the reliability of SCNT to produce healthy GM founder pigs for further studies. From 2006 to 2016, we worked with handmade cloning using donor skin cells from 4 breeds (2 minipigs, 2 standard pigs) that were non-GM or GM with 1 out of more than 20 genes. Cells were reconstructed with oocytes from Large White (LW) sows or gilts, and embryos were in vitro cultured for 5 to 6 days before selection for transfer to LW recipient sows or gilts (Callesen et al. 2014 Cell. Reprogram. 16, 407-410). Enough cloned embryos were produced with each type of GM donor cell for transfer to at least 2 recipients. During the first 4 years, the procedure was being established and refined, whereas in the last 7 years, it was used routinely (Table 1). In the latter period, the GM piglets alive after 30 days represented 17 of the 18 transgenes used. The GM piglets alive 30 days after birth were kept and developed as normal pigs. For 4 of the transgenes used, cloned minipigs were bred using standard breeding; in total, 106 piglets from 15 litters were born in the first generation after the cloning, and 138 piglets from 20 litters in the second generation. Both litter sizes and abnormality frequencies were within the expected range of the given breed (Yucatan or Göttingen), also noting that the pigs harbored a transgene and that some inbreeding was unavoidable due to the few founder piglets available. This work demonstrates that use of SCNT is a reliable way to produce GM founder piglets even though cloning does result in great losses during farrowing and the early postnatal period. However, having overcome these critical phases, the piglets seem to show no visible signs of their challenging background. The overall expense is, of course, high for production of each of the GM founder pigs, and this should be taken into consideration when deciding the species to use for creating a given GM animal model for modelling human diseases.

Table 1.  Results from 2010 to 2016 of using cloning to produce genetically modified (GM) and non-GM piglets