Steven R. Watt

Hyperacute rejection of porcine organs by old world primate recipients is mediated through preformed antibodies against galactosyl-alpha-1,3-galactose (Galalpha-1,3-Gal) epitopes expressed on the pig cell surface. Previously, we generated inbred miniature swine with a null allele of the alpha-1,3-galactosyltransferase locus (GGTA1) by nuclear transfer (NT) with gene-targeted fibroblasts. To expedite the generation of GGTA1 null pigs, we selected spontaneous null mutant cells from fibroblast cultures of heterozygous animals for use in another round of NT. An unexpectedly high rate of spontaneous loss of GGTA1 function was observed, with the vast majority of null cells resulting from loss of the WT allele. Healthy piglets, hemizygous and homozygous for the gene-targeted allele, were produced by NT by using fibroblasts that had undergone deletional and crossover/gene conversion events, respectively. Aside from loss of Galalpha-1,3-Gal epitopes, there were no obvious phenotypic differences between these null piglets and WT piglets from the same inbred lines. In fact, congenital abnormalities observed in the heterozygous NT animals did not reappear in the serially produced null animals.

Central to the success of large animal cloning is the production of healthy animals that can provide products for human health, food, and other animal agriculture applications. We report development of cloned cattle derived from 34 genetically unique, nonembryonic cell lines using nuclear transfer performed between 1 January 1998 and 29 February 2000. Nearly 25% (535/2170) of the recipients receiving reconstructed embryos initiated pregnancy. Overall, 19.8% (106/535) of the initiated pregnancies resulted in live births, while 77% (82/106) of these cattle clones remain healthy and productive today. Although a wide variation in birth weight of clone calves was observed, their growth rates, reproductive performance, and lactation characteristics are similar to that found in noncloned dairy cattle. Our data represent the most comprehensive information on cattle derived from nuclear transfer procedures and indicate that this emerging reproductive technology offers unique opportunities to meet critical needs in both human health care and agriculture.

Using an interwoven-loop experimental design in conjunction with highly conservative linear mixed model methodology using estimated variance components, 18 genes differentially expressed between nuclear transfer (NT)- and in vitro fertilization (IVF)-produced embryos were identified. The set is comprised of three intermediate-filament protein genes (cytokeratin 8, cytokeratin 19, and vimentin), three metabolic genes (phosphoribosyl pyrophosphate synthetase 1, mitochondrial acetoacetyl-coenzyme A thiolase, and alpha-glucosidase), two lysosomal-related genes (prosaposin and lysosomal-associated membrane protein 2), and a gene associated with stress responses (heat shock protein 27) along with major histocompatibility complex class I, nidogen 2, a putative transport protein, heterogeneous nuclear ribonuclear protein K, mitochondrial 16S rRNA, and ES1 (a zebrafish orthologue of unknown function). The three remaining genes are novel. To our knowledge, this is the first report comparing individual embryos produced by NT and IVF using cDNA microarray technology for any species, and it uses a rigorous experimental design that emphasizes statistical significance to identify differentially expressed genes between NT and IVF embryos in cattle.

The pregnancy initiation and maintenance rates of nuclear transfer embryos produced from several bovine cell types were measured to determine which cell types produced healthy calves and had growth characteristics that would allow for genetic manipulation. Considerable variability between cell types from one animal and the same cell type from different animals was observed. In general, cultured fetal cells performed better with respect to pregnancy initiation and calving than adult cells with the exception of cumulous cells, which produced the highest overall pregnancy and calving rates. The cell type that combined relatively high pregnancy initiation and calving rates with growth characteristics that allowed for extended proliferation in culture were fetal genital ridge (GR) cells. Cultured GR cells used in nuclear transfer and embryo transfer initiated pregnancies in 40% of recipient heifers (197), and of all recipients that received nuclear transfer embryos, 9% produced live calves. Cultured GR cells doubled as many as 85 times overall and up to 75 times after dilution to single-cell culture. A comparison between transfected and nontransfected cells showed that transfected cells had lower pregnancy initiation (22% versus 32%) and calving (3.4% versus 8.9%) rates.