Jose L. Estrada

Production of ELOVL4 transgenic pigs: a large animal model for Stargardt-like macular degeneration

Background Truncation mutations in the elongation of very long chain fatty acids-4 (AF277094, MIM #605512) (ELOVL4) gene cause Stargardt-like macular dystrophy type 3 (STGD3). Mice expressing truncated ELOVL4 develop rapid retinal degeneration, but are poor STGD3 models since mice lack a macula. Photoreceptor topography in the pig retina is more similar to that in humans as it includes the cone rich, macula-like area centralis. The authors generated transgenic pigs expressing human disease-causing ELOVL4 mutations to better model the pathobiology of this macular disease. Methods Pronuclear DNA microinjection and somatic cell nuclear transfer were used to produce transgenic pigs for two different ELOVL4 mutations: the 5 base pair deletion (5 bpdel) and the 270 stop mutation (Y270terEYFP). Retinal transgene expression, morphology and electrophysiology were examined. Results The authors obtained four lines of Y270terEYFP and one line of 5 bpdel transgenic animals. Direct fluorescence microscopy indicated that the Y270terEYFP protein is expressed in photoreceptors and mislocalised within the cell. Immunohistochemical examination of transgenic pigs showed photoreceptor loss and disorganised inner and outer segments. Electroretinography demonstrated diminished responses in both transgenic models. Conclusions These transgenic pigs provide unique animal models for examining macular degeneration and STGD3 pathogenesis.

Detection of transcriptional difference of porcine imprinted genes using different microarray platforms

BackgroundPresently, multiple options exist for conducting gene expression profiling studies in swine. In order to determine the performance of some of the existing microarrays, Affymetrix Porcine, Affymetrix Human U133+2.0, and the U.S. Pig Genome Coordination Program spotted glass oligonucleotide microarrays were compared for their reproducibility, coverage, platform independent and dependent sensitivity using fibroblast cell lines derived from control and parthenogenic porcine embryos.ResultsArray group correlations between technical replicates demonstrated comparable reproducibility in both Affymetrix arrays. Glass oligonucleotide arrays showed greater variability and, in addition, approximately 10% of probes had to be discarded due to slide printing defects. Probe level analysis of Affymetrix Human arrays revealed significant variability within probe sets due to the effects of cross-species hybridization. Affymetrix Porcine arrays identified the greatest number of differentially expressed genes amongst probes common to all arrays, a measure of platform sensitivity. Affymetrix Porcine arrays also identified the greatest number of differentially expressed known imprinted genes using all probes on each array, an ad hoc measure of realistic performance for this particular experiment.ConclusionWe conclude that of the platforms currently available and tested, the Affymetrix Porcine array is the most sensitive and reproducible microarray for swine genomic studies.

Successful Cloning of the Yucatan Minipig Using Commercial/Occidental Breeds as Oocyte Donors and Embryo Recipients

The widespread application of porcine SCNT to biomedical research is being hampered by the large adult size (300-600 lbs) of the commercial breeds commonly used for SCNT. The Yucatan minipig, in contrast, has an adult weight of 140-150 lbs and a long history of utility in biomedical research. In order to combine the wide availability of commercial swine with the biomedical value of the Yucatan minipig, we utilized SCNT using the Yucatan as nuclear donors and commercial swine as both oocyte donors and recipients. Of six recipient gilts receiving 631 SCNT embryos, three went to term and delivered seven piglets, four of which survived to adulthood. Additionally, we obtained fetal fibroblasts from a cloned Yucatan and used them for a second round of SCNT. Of three recipients receiving 315 reconstructed embryos, one went to term and delivered three piglets, one of which survived to adulthood. Both microsatellite and D-loop sequence analysis confirmed that all of the piglets generated were nuclear-mitochondrial hybrids carrying Yucatan nuclear DNA and commercial breed mitochondrial DNA. This report shows that it is possible to produce viable Yucatan SCNT clones and opens up the possibility of developing valuable biomedical models in this porcine breed.

A Comparative Study on the Efficiency of Two Enucleation Methods in Pig Somatic Cell Nuclear Transfer: Effects of the Squeezing and the Aspiration Methods

In this study, two enucleation methods, the squeezing and the aspiration methods, were compared. The efficiency of these two methods to enucleate pig oocytes and the in vitro and in vivo viability of somatic cell nuclear transfer (SCNT) pig embryos, were evaluated. In the squeezing method, the zona pellucida was partially dissected and a small amount of cytoplasm containing metaphase II (MII) chromosomes and the first polar body (PB) were pushed out. In the aspiration method, the PB and MII chromosomes were aspirated using a beveled micropipette. After injection of fetal fibroblasts into the perivitelline space, reconstructed oocytes were fused and activated electrically, and then cultured in vitro for 6 days or transferred to surrogates. The squeezing method resulted in a higher proportion of degenerated oocytes than the aspiration method (14% vs. 5%). The squeezing method took longer to enucleate 100 oocytes (306 minutes) than the aspirating method (113 minutes). Fusion rate (72–78%) and cleavage rate (67%) were not influenced by the enucleation method but blastocyst formation was improved (P < 0.05) in oocytes enucleated by the aspiration method (5 vs. 9%). When SCNT embryos were transferred to recipients, pregnancy rates to term were similar (27%, 3/11 and 27%, 3/11) in both methods with the birth of 10 piglets/3 litters and 16 piglets/3 litters in the squeezing and the aspiration methods, respectively. Our results indicate that the aspiration method for oocyte enucleation is more efficient than the squeezing method in producing a large number of pig SCNT embryos with normal in vivo viability.