Xenotransplantation from pigs could alleviate the shortage of human tissues and organs for transplantation. Means have been identified to overcome hyperacute rejection and acute vascular rejection mechanisms mounted by the recipient. The challenge is to combine multiple genetic modifications to enable normal animal breeding and meet the demand for transplants. We used two methods to colocate xenoprotective transgenes at one locus, sequential targeted transgene placement - ‘gene stacking’, and cointegration of multiple engineered large vectors - ‘combineering’, to generate pigs carrying modifications considered necessary to inhibit short to mid-term xenograft rejection. Pigs were generated by serial nuclear transfer and analysed at intermediate stages. Human complement inhibitors CD46, CD55 and CD59 were abundantly expressed in all tissues examined, human HO1 and human A20 were widely expressed. ZFN or CRISPR/Cas9 mediated homozygous GGTA1 and CMAH knockout abolished α-Gal and Neu5Gc epitopes. Cells from multi-transgenic piglets showed complete protection against human complement-mediated lysis, even before GGTA1 knockout. Blockade of endothelial activation reduced TNFα-induced E-selectin expression, IFNγ-induced MHC class-II upregulation and TNFα/cycloheximide caspase induction. Microbial analysis found no PERV-C, PCMV or 13 other infectious agents. These animals are a major advance towards clinical porcine xenotransplantation and demonstrate that livestock engineering has come of age.

Alterations in the nuclear positioning of chromosomes and specific genes during differentiation and development have suggested strongly the existence of a relationship between non-random organization of the genome and its function. In this study, we have examined the genome organization in interphase nuclei during adipogenesis, using the pig as a model organism. We hypothesized that changes in the gene expression profile and chromatin remodeling which occur during cellular differentiation would elicit repositioning of whole chromosomes, moving specific genes on them to different regions of the nucleus. We established an in vitro adipogenesis differentiation system using mesenchymal stem cells, derived from porcine bone marrow. The nuclear position of seven adipogenesis genes (PPARG, SREBF1, FABP4, CEBPA, CEBPB, CREB, and GATA2), two control genes (SOX9 and MYL1), and six chromosomes carrying these gene loci (SSC4, SSC6, SSC12, SSC13, SSC15, and SSC17) was determined. We found that during adipogenesis, using the in vitro stem cell model system, in contrast to our original hypothesis, the nuclear position of genes involved in adipogenesis was altered radically with the up-regulation of gene expression correlating with these genes becoming more internally located within nuclei. Chromosome territories, containing these genes, were also found to alter their nuclear position during the in vitro adipogenesis model, with the most dramatic repositioning being SSC4 that moved from the nuclear periphery towards the nuclear interior. We found that during in vitro adipogenesis chromosome territories decondensed and the genes were found on loops and projections of chromatin, away from the main body of the chromosomes. From our data, it appears that the temporal repositioning of genes, emanating away from chromosomes, during adipogenesis is correlated with gene activity, supporting models of the involvement of spatial genome repositioning in regulating gene expression and the nuclear interior being an important region of the nucleus for transcription.

Self-interacting chromatin domains encompass genes and their cis-regulatory elements; however, the three-dimensional form a domain takes, whether this relies on enhancer–promoter interactions, and the processes necessary to mediate the formation and maintenance of such domains, remain unclear. To examine these questions, here we use a combination of high-resolution chromosome conformation capture, a non-denaturing form of fluorescence in situ hybridisation and super-resolution imaging to study a 70 kb domain encompassing the mouse α-globin regulatory locus. We show that this region forms an erythroid-specific, decompacted, self-interacting domain, delimited by frequently apposed CTCF/cohesin binding sites early in terminal erythroid differentiation, and does not require transcriptional elongation for maintenance of the domain structure. Formation of this domain does not rely on interactions between the α-globin genes and their major enhancers, suggesting a transcription-independent mechanism for establishment of the domain. However, absence of the major enhancers does alter internal domain interactions. Formation of a loop domain therefore appears to be a mechanistic process that occurs irrespective of the specific interactions within.