Factors influencing the efficiency of generating genetically engineered pigs by nuclear transfer: multi-factorial analysis of a large data set

Abstract

BackgroundSomatic cell nuclear transfer (SCNT) using genetically engineered donor cells is currently the most widely used strategy to generate tailored pig models for biomedical research. Although this approach facilitates a similar spectrum of genetic modifications as in rodent models, the outcome in terms of live cloned piglets is quite variable. In this study, we aimed at a comprehensive analysis of environmental and experimental factors that are substantially influencing the efficiency of generating genetically engineered pigs. Based on a considerably large data set from 274 SCNT experiments (in total 18,649 reconstructed embryos transferred into 193 recipients), performed over a period of three years, we assessed the relative contribution of season, type of genetic modification, donor cell source, number of cloning rounds, and pre-selection of cloned embryos for early development to the cloning efficiency.Results109 (56%) recipients became pregnant and 85 (78%) of them gave birth to offspring. Out of 318 cloned piglets, 243 (76%) were alive, but only 97 (40%) were clinically healthy and showed normal development. The proportion of stillborn piglets was 24% (75/318), and another 31% (100/318) of the cloned piglets died soon after birth. The overall cloning efficiency, defined as the number of offspring born per SCNT embryos transferred, including only recipients that delivered, was 3.95%. SCNT experiments performed during winter using fetal fibroblasts or kidney cells after additive gene transfer resulted in the highest number of live and healthy offspring, while two or more rounds of cloning and nuclear transfer experiments performed during summer decreased the number of healthy offspring.ConclusionAlthough the effects of individual factors may be different between various laboratories, our results and analysis strategy will help to identify and optimize the factors, which are most critical to cloning success in programs aiming at the generation of genetically engineered pig models.

Efficient modification of the myostatin gene in porcine somatic cells and generation of knockout piglets

“…Here, we obtained six offspring: two (33.3%) were stillborn and three (50%) died within 24 hr after birth. Our stillbirth percentage was similar to the findings of a large‐scale study using more than 300 cloned piglets, which found that 24% were stillborn and 31% died soon after birth (Kurome et al, ). In this study, the piglets that died within 24 hr after birth weighed less at birth than wild‐type piglets.…”

Generation of Five Human Lactoferrin Transgenic Cloned Goats Using Fibroblast Cells and Their Methylation Status of Putative Differential Methylation Regions of IGF2R and H19 Imprinted Genes

“…Lived clones obtained from adult somatic cells in sheep [31], [32], cattle [33]-[36], and pigs [37]-[40] have also been reported. Kurome et al indicated that there was no significant difference for the effects of different donor cell sources on cloning success [41]. In the present study, we used fibroblast cells isolated from ear tissue of the two-month-old dairy goat as donor cells for SCNT.…”

Achievements and unmet promises of assisted reproduction technologies in large animals: a per-sonal perspective

“…In the pig the situation is intermediate and no dramatic phenotypes are reported (Kurome et al, 2013). The low efficiency is compensated by the transfer of many cloned embryos per recipients to ensure a high pregnancy rate and a reasonable number of newborn piglets.…”