Assembling multiple xenoprotective transgenes in pigs

Xenotransplantation is a promising strategy to alleviate the shortage of organs for human transplantation. In addition to the concern on pig-to-human immunological compatibility, the risk of cross-species transmission of porcine endogenous retroviruses (PERVs) has impeded the clinical application of this approach. Earlier, we demonstrated the feasibility of inactivating PERV activity in an immortalized pig cell line. Here, we confirmed that PERVs infect human cells, and observed the horizontal transfer of PERVs among human cells. Using CRISPR-Cas9, we inactivated all the PERVs in a porcine primary cell line and generated PERV-inactivated pigs via somatic cell nuclear transfer. Our study highlighted the value of PERV inactivation to prevent cross-species viral transmission and demonstrated the successful production of PERV-inactivated animals to address the safety concern in clinical xenotransplantation.

Integration, excision, and inversion of defined DNA segments commonly occur through site-specific recombination, a process of DNA breakage and reunion that requires no DNA synthesis or high-energy cofactor. Virtually all identified site-specific recombinases fall into one of just two families, the tyrosine recombinases and the serine recombinases, named after the amino acid residue that forms a covalent protein-DNA linkage in the reaction intermediate. Their recombination mechanisms are distinctly different. Tyrosine recombinases break and rejoin single strands in pairs to form a Holliday junction intermediate. By contrast, serine recombinases cut all strands in advance of strand exchange and religation. Many natural systems of site-specific recombination impose sophisticated regulatory mechanisms on the basic recombinational process to favor one particular outcome of recombination over another (for example, excision over inversion or deletion). Details of the site-specific recombination processes have been revealed by recent structural and biochemical studies of members of both families.

Highly efficient phage-based Escherichia coli homologous recombination systems have recently been developed that enable genomic DNA in bacterial artificial chromosomes to be modified and subcloned, without the need for restriction enzymes or DNA ligases. This new form of chromosome engineering, termed recombinogenic engineering or recombineering, is efficient and greatly decreases the time it takes to create transgenic mouse models by traditional means. Recombineering also facilitates many kinds of genomic experiment that have otherwise been difficult to carry out, and should enhance functional genomic studies by providing better mouse models and a more refined genetic analysis of the mouse genome.