Current progress of genetically engineered pig models for biomedical research

Abstract

The first transgenic pigs were generated for agricultural purposes about three decades ago. Since then, the micromanipulation techniques of pig oocytes and embryos expanded from pronuclear injection of foreign DNA to somatic cell nuclear transfer, intracytoplasmic sperm injection-mediated gene transfer, lentiviral transduction, and cytoplasmic injection. Mechanistically, the passive transgenesis approach based on random integration of foreign DNA was developed to active genetic engineering techniques based on the transient activity of ectopic enzymes, such as transposases, recombinases, and programmable nucleases. Whole-genome sequencing and annotation of advanced genome maps of the pig complemented these developments. The full implementation of these tools promises to immensely increase the efficiency and, in parallel, to reduce the costs for the generation of genetically engineered pigs. Today, the major application of genetically engineered pigs is found in the field of biomedical disease modeling. It is anticipated that genetically engineered pigs will increasingly be used in biomedical research, since this model shows several similarities to humans with regard to physiology, metabolism, genome organization, pathology, and aging.

Keywords: binary transposon; disease model; domestic animal; humanized pig; large animal model; programmable nuclease.

FIG. 1.

Transgenic pigs as models for human diseases. (A) Rodent models differ in a number of critical parameters, such as size, life span, genome organization, physiology, and metabolism, from humans. (B) The domesticated pig and its minipig varieties seem to be relevant models for specific human diseases.

FIG. 2.

Fluorescent reporter transposon pigs as tool for vital cell tracking. (A) Schematic depiction of Sleeping Beauty (SB)–Venus transposon. (B) Transposon transgenic pigs, generated by cytoplasmic injection of plasmids, carrying a Venus reporter fluorescent green under specific excitation. Wild-type animals (front) appear bluish due to reflected and scattered excitation light. (C) Amazingly, the Venus reporter is also deposited in the hair, and two samples from transgenic (left and middle) and a hair sample from a wild-type pig (right) are shown under epifluorescence. (D) Same view as in (C) showing under darkfield conditions. Scale bar=1 cm. (E) Spermatozoa from a reporter transposon boar shown under differential interference contrast (DIC) and (F) as overlay of specific fluorescence and DIC images. The spermatozoa loaded with the Venus protein and allow tracking the fate of paternal proteins after fertilization. Scale bar=5 μm. (G) Porcine induced pluripotent stem cell colonies derived from an SB–Venus animal shown under Venus fluorescence conditions, and (H) brightfield view.

FIG. 3.

Genetic engineering of the pig genome. (A) Additive genome engineering by transposase-catalyzed integration. Subsequently, a targeted replacement of the reporter against a transgene of choice is possible via Cre recombinase-mediated cassette exchange (RMCE).^, (B) Subtractive genome engineering by a programmable nuclease approach. A designer nuclease, ZFN, TALEN, or CRISPR/Cas9, is directed against a protein-coding exon. The CRISPR/Cas9 system seems to combine the efficiency of ZFNs and TALENs with a much simpler design principle, as target-site selection is determined solely by base complementarity to the guide RNA. Indel formation after faulty repair will likely result in a frameshift mutation and a functional gene knockout. In principle, this mechanism can be coupled with homologous recombination.