Genes, BACs and artificial chromosomes

Abstract

Xenotransplantation could alleviate the serious shortage of human donor organs and tissues, but clinically acceptable donor pigs require a complex battery of genetic modifications. The precise requirements continue to be refined as knowledge increases, but a current ‘wish list’ includes removal of Gal and non-Gal surface antigens, expression of complement regulators, anti-thrombotic or anticoagulant factors, vascular protection and immunomodulatory genes, and some means of preventing possible zoonotic infection. Several pig lines have already been produced for xenotransplantation. Other than α1,3GT gene-targeted animals, all contain individual randomly integrated transgenes. Transgenes integrated at different sites can however exhibit wide variations in the level and pattern of expression due to the influence of different chromatin environments and endogenous regulatory elements. The “position variegation effect” has been a long-standing source of inefficiency and contributes to the inadequate expression observed for example in human complement regulator transgenes CD46, CD55 and CD59. This is exacerbated by the structure of traditional transgene expression vectors. In particular, viral promoters, small promoters, and cDNA or minigene rather than genomic sequences, are not reliable means of obtaining abundant uniform expression in transgenic animals. Novel approaches are therefore required to produce complex multi-transgenic animals. Fortunately a broad range of new technologies are becoming available that significantly increase the chances of success. For example, construction of large transgenes has been aided by the use of BACs for molecular cloning and in vivo recombination (recombineering) in bacteria. The position effect can be overcome by incorporating elements such as nuclear matrix attachment regions, or employing episomal vectors such as mammalian artificial chromosomes. Placement of transgenes at predetermined favourable sites by a variety of recombination systems has become routine in the mouse and will soon be extended to pigs and other livestock. Viral- and transposon based vectors greatly improve the efficiency in obtaining transgenic animals. Although size-restricted these are ideal for gene knockdown by short hairpin or tissue specific micro RNAs. Gene targeting became a reality in livestock species more than a decade ago with the advent of somatic cell nuclear transfer, but remains more difficult than in mouse ES cells. Promoter-trap or large BAC based vectors offer incremental improvements, but new tools, particularly meganucleases and zinc finger nucleases (ZFNs) are set to revolutionise the field. ZFNs allow gene knockout directly in a fertilised oocyte, circumventing the need for cultured cells and nuclear transfer. More interestingly it has recently been shown that ZFNs can facilitate other forms of gene targeting such as gene replacement in the oocyte. Approaches that still rely on cell-mediated transgenesis are also set to gain new possibilities with the announcement of pigs derived from induced pluripotent stem cells. Finally, the abundance of DNA sequence information, including the porcine genome due to be released in 2010, provides a vital resource. The cost of whole genome sequencing has decreased so dramatically (approaching $3000), that regulatory authorities can easily be provided with the complete sequence of the “ultimate xeno-pig” no matter what methods have been used to derive it.