Nucleofection-mediated α1,3-galactosyltransferase gene inactivation and membrane cofactor protein expression for pig-to-primate xenotransplantation.

Xenotransplantation of pig organs into primates leads to hyperacute rejection (HAR). Functional ablation of the pig α 1,3-galactosyltransferase (GalT) gene, which abrogates expression of the Gal α 1-3Gal ß 1-4GlcNAc-R (Gal) antigen, which inhibits HAR. However, antigens other than Gal may induce immunological rejection by their cognate antibody responses. Ultimately, overexpression of complement regulatory proteins reduces acute humoral rejection by non-Gal antibodies when GalT is ablated. In this study, we developed a vector-based strategy for ablation of GalT function and concurrent expression of membrane cofactor protein (MCP, CD46). We constructed an MCP expression cassette (designated as MCP-IRESneo) and inserted between the left and the right homologous arms to target exon 9 of the GalT gene. Nucleofection of porcine ear skin fibroblasts using the U-023 and V-013 programs resulted in high transfection efficiency and cell survival. We identified 28 clones in which the MCP-IRESneo vector had been successfully targeted to exon 9 of the GalT gene. Two of those clones, with apparent morphologically mitotic fibroblast features were selected through long-term culture. GalT gene expression was downregulated in these 2 clones. Importantly, MCP was shown to be efficiently expressed at the cell surface and to efficiently protect cell lysis against normal human complement serum attack in vitro.

Cortical gyrification and its relationships with molecular measures and cognition in children with the FMR1 premutation.

Neurobiological basis for cognitive development and psychiatric conditions remains unexplored in children with the FMR1 premutation (PM). Knock-in mouse models of PM revealed defects in embryonic cortical development that may affect cortical folding. Cortical-folding complexity quantified using local gyrification index (LGI) was examined in 61 children (age 8-12 years, 19/14 male/female PM carriers, 15/13 male/female controls). Whole-brain vertex-wise analysis of LGI was performed for group comparisons and correlations with IQ. Individuals with aberrant gyrification in 68 cortical areas were identified using Z-scores of LGI (hyper: Z ≥ 2.58, hypo: Z ≤ - 2.58). Significant group-by-sex-by-age interaction in LGI was detected in right inferior temporal and fusiform cortices, which correlated negatively with CGG repeat length in the PM carriers. Sixteen PM boys (hyper/hypo: 7/9) and 10 PM girls (hyper/hypo: 2/5, 3 both) displayed aberrant LGI in 1-17 regions/person while 2 control boys (hyper/hypo: 0/2) and 2 control girls (hyper/hypo: 1/1) met the same criteria in only 1 region/person. LGI in the precuneus and cingulate cortices correlated positively with IQ scores in PM and control boys while negatively in PM girls and no significant correlation in control girls. These findings reveal aberrant gyrification, which may underlie cognitive performance in children with the PM.