Effects of contaminant exposure on reproductive success of ospreys (Pandion haliaetus) nesting in Delaware River and Bay, USA.

Despite serious water-quality problems and pollutant loading and retention, Delaware River and Bay (USA) provide important wildlife habitat. In 2002, we conducted a comprehensive evaluation of contaminant exposure and reproduction of ospreys (Pandion haliaetus) breeding in Delaware River and Bay. Sample eggs were collected from 39 nests and analyzed for organochlorine pesticides, polychlorinated biphenyls (PCBs), and mercury; a subset of 15 eggs was analyzed for perfluorinated compounds and polybrominated diphenyl ethers (PBDEs). The fate of each nest was monitored weekly. Concentrations of 10 organochlorine pesticides or metabolites, total PCBs, and several toxic PCB congeners were greater (p < 0.05) in eggs collected between the Chesapeake and Delaware Canal (C and D Canal) and Trenton (Delaware River and northern Bay) compared to other sites. Concentrations of p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE; 0.785-3.84 microg/g wet wt) and total PCBs (5.50-14.5 microg/g wet wt) in eggs collected between the C and D Canal and Trenton were similar to levels recently found in the Chesapeake Bay. In all study segments, at least one young fledged from 66 to 75% of nests. Productivity for Delaware Inland Bays (reference area) and southern Delaware Bay was 1.17 and 1.42 fledglings/active nest, respectively; north of the C and D Canal, productivity was 1.00 fledgling/active nest, which is marginally adequate to maintain the population. Using these data, a logistic regression model found that contaminant concentrations (p,p'-DDE, heptachlor epoxide, chlordane and metabolites, and total PCBs) were predictive of hatching success. Several perfluorinated compounds and PBDEs were detected in eggs at concentrations approaching 1 microg/g wet weight. These findings provide evidence that contaminants continue to be a significant stressor on osprey productivity in the northern Delaware River and Bay.

Production of alpha-1,3-galactosyltransferase null pigs by means of nuclear transfer with fibroblasts bearing loss of heterozygosity mutations.

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.

Transgenic pig expressing the enhanced green fluorescent protein produced by nuclear transfer using colchicine-treated fibroblasts as donor cells.

Fetal-derived fibroblast cells were transduced with replication defective vectors containing the enhanced green fluorescent protein (EGFP). The transgenic cells were treated with colchicine, which theoretically would synchronize the cells into G2/M stage, and then used as donor nuclei for nuclear transfer. The donor cells were transferred into the perivitalline space of enucleated in vitro matured porcine oocytes, and fused and activated with electrical pulses. A total of 8.3% and 28.6% of reconstructed oocytes showed nuclear envelope breakdown and premature chromosome condensation 0.5 and 2 hr after activation, respectively. Percentage of pronuclear formation was 62.5, 12 hr after activation. Most (91.4%) of the 1-cell embryos with pronuclei did not extrude a polar body. Most (77.2%) embryos on day 5 were diploid. Within 2 hr after fusion, strong fluorescence was detectable in most reconstructed oocytes (92.3%). The fluorescence in all NT embryos became weak 15 hr after fusion and disappeared when culture to 48 hr. But from day 3, cleaved embryos at the 2- to 4-cell stage started to express EGFP again. On day 7, 85.8% of cleaved embryos expressed EGFP. A total of 9.4% of reconstructed embryos developed to blastocyst stage and 71.5% of the blastoctysts expressed EGFP. After 200 reconstructed 1-cell stage embryos were transferred into four surrogate gilts, three recipients were found to be pregnant. One of them maintained to term and delivered a healthy transgenic piglet expressing EGFP. Our data suggest that the combination of transduction of somatic cells by a replication defective vector with the nuclear transfer of colchicine-treated donors is an alternative to produce transgenic pigs. Furthermore, the tissues expressing EGFP from descendents of this pig may be very useful in future studies using pigs that require genetically marked cells.

Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning.

The presence of galactose alpha-1,3-galactose residues on the surface of pig cells is a major obstacle to successful xenotransplantation. Here, we report the production of four live pigs in which one allele of the alpha-1,3-galactosyltransferase locus has been knocked out. These pigs were produced by nuclear transfer technology; clonal fetal fibroblast cell lines were used as nuclear donors for embryos reconstructed with enucleated pig oocytes.