Retinitis pigmentosa (RP) is a prevalent cause of blindness caused by a large number of different mutations in many different genes. The mutations result in rod photoreceptor cell death, but it is unknown why cones die. In this study, we tested the hypothesis that cones die from oxidative damage by performing immunohistochemical staining for biomarkers of oxidative damage in a transgenic pig model of RP. The presence of acrolein- and 4-hydroxynonenal-adducts on proteins is a specific indicator that lipid peroxidation has occurred, and there was strong immunofluorescent staining for both in cone inner segments (IS) of two 10-month-old transgenic pigs in which almost all rods had died, compared to faint staining in two 10-month-old control pig retinas. In 22- and 24-month-old transgenic pigs in which all rods and many cones had died, staining was strong in cone axons and some cell bodies as well as IS indicating progression in oxidative damage between 10 and 22 months. Biomarkers for oxidative damage to proteins and DNA also showed progressive oxidative damage to those macromolecules in cones during the course of RP. These data support the hypothesis that the death of rods results in decreased oxygen consumption and hyperoxia in the outer retina resulting in gradual cone cell death from oxidative damage. This hypothesis has important therapeutic implications and deserves rapid evaluation.

In addition to rod photoreceptor loss, many mutations in rod photoreceptor-specific genes cause degeneration of other neuronal types. Identifying mechanisms of cell-cell interactions initiated by rod-specific mutations and affecting other retinal cells is important for understanding the pathogenesis and progression of retinal degeneration. Here we show in animals with rod and cone degeneration due to mutations in the genes encoding rhodopsin and cGMP phosphodiesterase beta-subunit (PDE-beta) respectively, that rod bipolar cells received ectopic synapses from cones in the absence of rods. Thus, synaptic plasticity links certain rod-specific mutations to retina-wide structural alterations that involve different types of neurons.

Pig embryos at the 1- or 2-cell stage (before the 'block' to development in vitro) were cultured in 8 different media derived from Krebs'-Ringer-bicarbonate medium. A 2 x 2 x 2 factorial arrangement was used for the treatments, with glucose, glutamine and phosphate being the major effects tested. Embryos were obtained from sows approximately 44-48 h after the observation of oestrus, with the majority being at the 1-cell stage. Embryos from each female were randomly assigned to each treatment. After in-vitro culture, all embryos were scored for the stage of development attained and stained to determine final cell number. Significant effects were evident due to female, glucose, glutamine, a phosphate x glucose interaction and a glutamine x glucose interaction. None of the media components tested was inhibitory to embryo development. The greatest development (45-60% morula or blastocyst) was achieved with glucose and glutamine (both alone and in combination) in the media, demonstrating that an amino acid can serve as the sole energy source for complete preimplantation embryonic development in vitro.

Photoreceptors of transgenic mice expressing a mutant rhodopsin gene (Prom7?+ Ser) slowly degenerate. The mechanism of degeneration was studied by aggregation of embryos of normal and transgenic mice to form chimeras. In these chimeras, mosaicism was observed in the coat color, retinal pigment epithelium, and retina. In the retina, the genotype of adjacent patches of normal and transgenic photoreceptors was determined by in situ hybridization with a transgene-speciffc RNA probe. (13). The two strains employed, a transgenic mouse line hemizygous for a Pro347 -_+Ser mutation in the pig rhodopsin gene and a normal strain, shared the same genetic background, C57BL/6. The transgenic strain was constructed by using C57BL/6 mice and maintained by crossing hemizygous individuals with C57BL/6 mice. Potentially transgenic embryos were generated by mating hemizygous transgenic male mice to C57BL/6 female mice. As a result, half of the embryos should be transgenic. The normal mice, C57BL/6J -c2J/c2J (The Jackson Laboratory), were homozygous for albino, allowing identification of chimeras by coat color mosaicism and by observation of mosaic fundi with indirect ophthalmoscopy. Chimeras expressing the transgene were identified by standard tail-DNA analysis.Histology. Three chimeras expressing the transgene were sacrificed at 7 weeks of age, and the one lacking the transgene, at 14 weeks. Enucleated eyes were immersed in 0.1 M cacodylate buffer, pH 7.3/3% glutaraldehyde. Following overnight fixation at 4°C, eyes were bisected through the optic nerve head along either a superior-inferior or nasaltemporal axis. The eyes were postfixed in 2% osmium tetroxide and embedded in low-viscosity Spurr resin (14). Sections included the entire hemisphere that passed near the optic nerve head. Sections 0.5-1 ,um thick were taken and counterstained with toluidine blue dye.Construction and Synthesis of Probes for in Situ Hybridization. Two RNA probes were used for in situ hybridization; one specific for mouse rhodopsin and the other, pig rhodopsin. These probes were derived from DNA sequences in the 3' untranslated portions of the respective rhodopsin genes. The DNA fragments were cloned in the Novagen T-vector

Pig embryos can be cultured using a number of different strategies including complex approaches like culture in vivo in a surrogate oviduct (rabbit, sheep, mouse), culture in mouse oviducts in organ culture, and co-culture of embryos with cells in addition to simple approaches like culture in defined media or salt solutions.Addition of serum to medium has been of particular importance where blastocyst development and hatching are required. Pig conceptuses (day 10-15), embryonic discs or cell lines derived from conceptuses can be cultured in complex media like Eagle's minimal essential medium or Dulbecco's modified Eagle's medium with serum, although embryonic discs can be cultured in the absence of serum. In contrast, early stage pig embryos (one-cell to blastocyst) are best cultured in simpler media such as those used for mouse embryos. The media that have been used are all relatively similar in composition.They contain salts and one or more energy sources such as glucose, lactate, or pyruvate with BSA as a macromolecular component. Early attempts to culture pig embryos were not very successful, but some embryos did develop to the blastocyst stage. More recent reports indicate a much higher rate of development with relatively little or no change in media composition. Some workers have reported improved development in medium lacking glucose, which is consistent with findings with laboratory animals such as hamsters. Glutamine can serve as the sole exogenous energy source in medium lacking glucose, lactate and pyruvate.Addition of taurine and hypotaurine to culture medium enhances development of pig embryos in vitro. We suggest, where possible, adoption of a standard medium that could be used by different laboratories and, perhaps, with different species. Use of one medium for different species would simplify experimental protocols, enhance studies of comparative embryonic physiology and metabolism, and expedite transfer of information obtained in different species.completely defined (Petters, 1992). Furthermore, the culture requirements of pig embryos at different stages may be distinctive and require special solutions. Applications in basic research and in biotechnology will benefit from the increased embryo viability associated with improved culture methods. This review documents current methods for pig embryo culture and indicates areas where further research is warranted. Other aspects are covered in Reed et al. (1992a).