Search

Infectious diseases of farmed and wild animals pose a recurrent threat to food security and human health. The macrophage, a key component of the innate immune system, is the first line of defence against many infectious agents and plays a major role in shaping the adaptive immune response. However, this phagocyte is a target and host for many pathogens. Understanding the molecular basis of interactions between macrophages and pathogens is therefore crucial for the development of effective strategies to combat important infectious diseases. We explored how porcine pluripotent stem cells (PSCs) can provide a limitless in vitro supply of genetically and experimentally tractable macrophages. Porcine PSC-derived macrophages (PSCdMs) exhibited molecular and functional characteristics of ex vivo primary macrophages and were productively infected by pig pathogens, including porcine reproductive and respiratory syndrome virus (PRRSV) and African swine fever virus (ASFV), two of the most economically important and devastating viruses in pig farming. Moreover, porcine PSCdMs were readily amenable to genetic modification by CRISPR/Cas9 gene editing applied either in parental stem cells or directly in the macrophages by lentiviral vector transduction. We show that porcine PSCdMs exhibit key macrophage characteristics, including infection by a range of commercially relevant pig pathogens. In addition, genetic engineering of PSCs and PSCdMs affords new opportunities for functional analysis of macrophage biology in an important livestock species. PSCs and differentiated derivatives should therefore represent a useful and ethical experimental platform to investigate the genetic and molecular basis of host-pathogen interactions in pigs, and also have wider applications in livestock.

Background: Pig‐derived tissues could overcome the shortage of human donor organs in transplantation. However, the glycans with terminal α‐Gal and Neu5Gc, which are synthesized by enzymes, encoded by the genes GGTA1 and CMAH, are known to play a major role in immunogenicity of porcine tissue, ultimately leading to xenograft rejection. Methods: The N‐glycome and glycosphingolipidome of native and decellularized porcine pericardia from wildtype (WT), GGTA1‐KO and GGTA1/CMAH‐KO pigs were analyzed by multiplexed capillary gel electrophoresis coupled to laser‐induced fluorescence detection. Results: We identified biantennary and core‐fucosylated N‐glycans terminating with immunogenic α‐Gal‐ and α‐Gal‐/Neu5Gc‐epitopes on pericardium of WT pigs that were absent in GGTA1 and GGTA1/CMAH‐KO pigs, respectively. Levels of N‐glycans terminating with galactose bound in β(1‐4)‐linkage to N‐acetylglucosamine and their derivatives elongated by Neu5Ac were increased in both KO groups. N‐glycans capped with Neu5Gc were increased in GGTA1‐KO pigs compared to WT, but were not detected in GGTA1/CMAH‐KO pigs. Similarly, the ganglioside Neu5Gc‐GM3 was found in WT and GGTA1‐KO but not in GGTA1/CMAH‐KO pigs. The applied detergent based decellularization efficiently removed GSL glycans. Conclusion: Genetic deletion of GGTA1 or GGTA1/CMAH removes specific epitopes providing a more human‐like glycosylation pattern, but at the same time changes distribution and levels of other porcine glycans that are potentially immunogenic. [ABSTRACT FROM AUTHOR]

The sex-determining region on the Y chromosome (SRY) is thought to be the central genetic element of male sex development in mammals. Pathogenic modifications within the SRY gene are associated with a male-to-female sex reversal syndrome in humans and other mammalian species, including rabbits and mice. However, the underlying mechanisms are largely unknown. To understand the biological function of the SRY gene, a site-directed mutational analysis is required to investigate associated phenotypic changes at the molecular, cellular, and morphological level. Here, we successfully generated a knockout of the porcine SRY gene by microinjection of two CRISPR-Cas ribonucleoproteins, targeting the centrally located "high mobility group" (HMG), followed by a frameshift mutation of the downstream SRY sequence. This resulted in the development of genetically male (XY) pigs with complete external and internal female genitalia, which, however, were significantly smaller than in 9-mo-old age-matched control females. Quantitative digital PCR analysis revealed a duplication of the SRY locus in Landrace pigs similar to the known palindromic duplication in Duroc breeds. Our study demonstrates the central role of the HMG domain in the SRY gene in male porcine sex determination. This proof-of-principle study could assist in solving the problem of sex preference in agriculture to improve animal welfare. Moreover, it establishes a large animal model that is more comparable to humans with regard to genetics, physiology, and anatomy, which is pivotal for longitudinal studies to unravel mammalian sex determination and relevant for the development of new interventions for human sex development disorders. [ABSTRACT FROM AUTHOR]

Background: The present study reports the development of a sensitive dot blot protocol for determining the level of preformed antibodies against porcine heart valve tissue derived from wild‐type (WT) and α‐Gal‐KO (GGTA1‐KO) pigs in human sera. Methods: The assay uses decellularized and solubilized heart valve tissue; antibody binding found in this dot blot assay could be correlated with antibody titers of preformed anti‐α‐Gal and anti‐Neu5Gc antibodies detected by a sensitive ELISA. Results: The ultimate protocol had an inter‐assay variance of 9.5% and an intra‐assay variance of 9.2%, showing that the test is reliable and highly reproducible. With the aid of this dot blot assay, we found significant variation with regard to antibody contents among twelve human sera. Binding of preformed antibodies to WT tissue was significantly higher than to GGTA1‐KO tissue. Conclusions: The dot blot assay described herein could be a valuable tool to measure preformed antibody levels in human sera against unknown epitopes on decellularized tissue prior to implantation. Ultimately, this prescreening may allow a matching of the porcine xenograft with the respective human recipients in demand and thus may become an important tool for graft long‐term survival similar to current allotransplantation settings. [ABSTRACT FROM AUTHOR]