Swine clones: potential application for animal production and animal models

Abstract

Somatic cell nuclear transfer (SCNT), or cloning, is used to reprogram cells and generate genetically identical embryos and animals. However, the cloning process is inefficient, limiting its application to producing valuable animals. In swine, cloning is mainly utilized to produce genetically modified animals. Indeed, recombinant DNA technologies have evolved considerably in recent years, with homologous recombination and gene editing technologies becoming more efficient and capable of recombining both alleles in a single cell. The selection of appropriate cells and their use as nuclear donors for SCNT is the most common method for generating edited and genetically modified animals for commercial and research purposes. This article reviews current applications of swine cloning and shares our personal experiences with the procedure in this species.

Keywords: SCNT; cloning; embryos; genetically modified swine; oocytes.

Figure 1. Illustration of the enucleation process for swine cloning using an inverted microscope equipped with a micromanipulation apparatus. (A) The matured pig oocyte is held in place by suction with the holding pipet (left side), while the injection pipet (right side) is positioned close to the first polar body; (B) Aspiration of the first polar body and adjacent ooplasm containing the metaphase plate without fluorescent DNA stain (Unpublished data).

Figure 2. Echography images of a SCNT pregnancy. (A) Image of a transabdominal echography showing a gestational vesicle of cloned minipig at 33 days after ET (April 05, 2023), and (B) a formed cloned fetus at 62 days after ET (May 04, 2023) (Unpublished data).

Figure 3. Somatic cell donor animal and cloned fetus. (A) Image of the original minipig from which donor cells were obtained (~1 month old); (B) Image of the stillborn cloned minipig conceptus on the day of cesarian section (at 114 days of gestation) obtained after 140 SCNT embryos and 22 parthenotes ET, distributed in both oviducts. The stillborn clone measured 196.84 mm crown-rump and weighed 320g (Unpublished data).

Figure 4. Macroscopic and histologic images of the cloned minipig lungs and heart showing apparently normal morphology. Macroscopic image of the lungs (A) and heart (D). Histologic images of the lungs stained with Hematoxylin and Eosin (B) and Masson stain (C). Histologic images of the heart stained with Hematoxylin and Eosin (E) and Masson stain (F). Images were acquired at 10x magnification (Unpublished data).

Figure 5. Number of research articles obtained in the Clarivate™ (Web of Science™) database in the last 11 years (2012-2023) related to genetic modifications in swine. The search was done with the keywords: “transgenic pigs,” “edited pigs,” and “pig xenotransplantation”. Overall, the number of research articles containing the keyword “transgenic pigs” decreased, while the number of publications mentioning “edited pigs” increased, possibly because of the CRISPR/cas9 technique efficiency. However, the keyword “pig xenotransplantation” did not change substantially, indicating a shift in the methodology for producing GM pigs for organ donation.