Generation of a Stable Transgenic Swine Model Expressing a Porcine Histone 2B-eGFP Fusion Protein for Cell Tracking and Chromosome Dynamics Studies.

Transgenic pigs have become an attractive research model in the field of translational research, regenerative medicine, and stem cell therapy due to their anatomic, genetic and physiological similarities with humans. The development of fluorescent proteins as molecular tags has allowed investigators to track cell migration and engraftment levels after transplantation. Here we describe the development of two transgenic pig models via SCNT expressing a fusion protein composed of eGFP and porcine Histone 2B (pH2B).

Nr4A1 mRNA regulation of FSH-induced meiotic resumption in bovine cumulus-oocyte complexes cultured in vitro.

Objective: 1) further define the time course required for transcription initiation in bovine cumulus-oocyte-complexes (COC); 2) determine the pattern of expression for Nr4A1 and Egr1 mRNAs in bovine COC; and, 3) reduce Nr4A1 mRNA expression using small interfering RNAs (siRNA) to determine the effect on breakdown of the germinal vesicle (GVBD).

Methods: A series of experiments were performed to define the time required for transcription initiation during FSH-induced maturation in bovine COCs, determine the pattern of expression for candidate mRNAs during GVBD, and use RNAi to determine their potential role in GVBD by examining whether candidate-specific siRNA can reduce mRNA expression in bovine COCs and affect the occurrence of GVBD.

Results: Transcription required for GVBD in bovine COC occurred as early as 30 min after culture initiation.

Photoreceptor Differentiation following Transplantation of Allogeneic Retinal Progenitor Cells to the Dystrophic Rhodopsin Pro347Leu Transgenic Pig.

Purpose. Transplantation of stem, progenitor, or precursor cells has resulted in photoreceptor replacement and evidence of functional efficacy in rodent models of retinal degeneration. Ongoing work has been directed toward the replication of these results in a large animal model, namely, the pig.

Cloning, characterization, and expression analysis of the pig (Sus scrofa) C1q tumor necrosis factor-related protein-5 gene.

Purpose: Autosomal dominant early-onset long anterior zonules (LAZs) and late-onset retinal degeneration (L-ORD) in humans are associated with the S163R mutation of the complement 1q-tumor necrosis factor related protein-5 (CTRP5) gene. For using the pig as an L-ORD model for the study of pathology, we cloned, characterized, and studied the expression profile of pig CTRP5 (pCTRP5).

Methods: The pCTRP5 was cloned and sequenced from porcine genomic DNA.

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.

Transgenic Stra8-EYFP pigs: a model for developing male germ cell technologies.

The male germ line in mammals is composed of self-renewing cells, spermatogonia, the meiotic spermatocytes and spermiogenic spermatids. Identification of these cell stages in vitro has been problematic. Transgenic animals expressing a marker gene with a promoter specific to certain cell stages in the testis would be a useful approach to identifying these cells in a viable state.

Phenotypic stability of Pro347Leu rhodopsin transgenic pigs as indicated by photoreceptor cell degeneration.

Rhodopsin (Pro347Leu) transgenic pigs are recognized to be an excellent model for the human disease, retinitis pigmentosa. First published in 1997, the rhodopsin transgenic pigs have been maintained since that time at North Carolina State University by outcrossing hemizygous boars to unrelated sows. Nine generations of outcrossing have been completed.

Identification of a promoter for the human C1Q-tumor necrosis factor-related protein-5 gene associated with late-onset retinal degeneration.

Purpose: The Complement-1q tumor necrosis factor-related protein 5 (C1QTNF5/CTRP5) gene is located in the 3' untranslated region of the Membrane Frizzled Related Protein (MFRP) gene, and these two genes are reported to be dicistronic. The authors examined the 5' upstream sequence of CTRP5 for the presence of a promoter regulating the expression of this gene.

Methods: The sequence upstream of the translational start site of human CTRP5 (hCTRP5) was analyzed by Promoter Inspector software.

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.

Multifocal electroretinogram in rhodopsin P347L transgenic pigs.

Purpose: Neural ectopic rewiring in retinal degeneration such as retinitis pigmentosa (RP) may form functional synapses between cones and rod bipolar cells that cause atypical signal processing. In this study, the multifocal electroretinograms (mfERGs) of a large animal model of RP, the rhodopsin P347L transgenic (Tg) pig, were measured to examine the sources and nature of altered signal processing.

Methods: mfERG responses from a 6-week-old Tg pig were recorded before and after sequential application of tetrodotoxin (TTX), N-methyl-D-aspartate (NMDA), 2-amino-4-phosphonobutyric acid (APB), and cis-2,3-piperidinedicarboylic acid (PDA), to identify contributions to the retinal signal from inner retinal neurons, the ON-pathway, the OFF-pathway, and photoreceptors.

Swine generated by somatic cell nuclear transfer have increased incidence of intrauterine growth restriction (IUGR).

While somatic cell nuclear transfer (SCNT) has been successful in several species, many pregnancies are lost and anomalies are found in fetal and perinatal stages. In this study SCNT and artificial inseminations (AI) populations were compared for litter size, average birth weight, piglets alive at birth, stillborn, mummies, dead at the first week, intrauterine growth restriction (IUGR) and large for gestational age (LGA). Twenty-three SCNT litters (143 individuals) were compared to 112 AI litters (1300 individuals).

Synchronization and superovulation of mature cycling gilts for the collection of pronuclear stage embryos.

An efficient protocol was developed to synchronize and superovulate mature pigs for the collection of pronuclear stage embryos suitable for DNA microinjection. A timed and coordinated regimen of Lutalyse, PG600 and Chorulon along with daily checking for estrus allowed synchronization of groups of gilts having estrous cycles at regular intervals. Pigs 10-16 days after the beginning of standing estrus have been successfully synchronized into estrus using this protocol.

Long-term neuroretinal full-thickness transplants in a large animal model of severe retinitis pigmentosa.

Background: The purpose of this study was to explore neuroretinal transplantation in a large animal model of severe retinitis pigmentosa and to establish graft development, long-term survival, graft-host integration, and effects on the host retina.

Methods: Rhodopsin transgenic pigs, aged 6 months, received in one eye a fetal full-thickness neuroretinal sheet in the subretinal space by means of vitrectomy and retinotomy. Six months postoperatively, eyes were studied in the light microscope and with immunohistochemical markers.

Reporter system for the detection of in vivo gene conversion: changing colors from blue to green using GFP variants.

We have devised a system for the study of in vivo gene correction based on the detection of color variants of the green fluorescent protein (GFP) from the jellyfish Aequorea victoria. The intensity and spectra of the fluorescence emitted by the blue (BFP) and red-shifted (EGFP) variants of GFP differ from each other. We modified one nucleotide from an EGFP expression vector that we predicted would yield a blue variant (TAC-CAC, Tyr(66)-His(66)).

Altered light responses of single rod photoreceptors in transgenic pigs expressing P347L or P347S rhodopsin.

Purpose: Numerous mutations of rhodopsin lead to rod cell death and ultimately to complete blindness, yet little is known about the alterations in the physiology of the light sensors containing the aberrant protein, the rod photoreceptors.

Methods: Suction pipettes were used to record the light responses from single rod photoreceptors isolated from the retinas of transgenic pigs of various ages and at progressive stages of retinal degeneration.

Results: We have observed changes in the photoresponse of transgenic porcine rods containing both wild type and mutant rhodospin.

Conditional induction of ovulation in mice.

Follicle-stimulating hormone controls the maturation of mammalian ovarian follicles. In excess, it can increase ovulation (egg production). Reported here is a transgenic doxycycline-activated switch, tested in mice, that produced more FSHB subunit (therefore more FSH) and increased ovulation by the simple feeding of doxycycline (Dox).

Oxidative damage is a potential cause of cone cell death in retinitis pigmentosa.

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.

Transplantation of full-thickness retina in the rhodopsin transgenic pig.

Purpose: To establish the morphology of full-thickness neuroretinal grafts transplanted to hosts with degenerative photoreceptor disease.

Methods: Twenty rhodopsin transgenic pigs received a neuroretinal sheet from a neonatal normal pig in one eye. Following vitrectomy and retinotomy with bleb formation, the grafts were positioned inside the bleb between the host neuroretina and retinal pigment epithelium.

Lensectomy and vitrectomy decrease the rate of photoreceptor loss in rhodopsin P347L transgenic pigs.

Background: Photoreceptor degeneration in retinitis pigmentosa (RP) runs an inevitable, gradually progressive course. A wide variety of growth factors of different origins have been shown to slow the rate of degeneration in some rodent models of RP. Recently, lens-derived neurotrophic factors have been shown to rescue degenerating ganglion cells in crush models of the optic nerve.

Roles of gene transcription and PKA subtype activation in maturation of murine oocytes.

The aims of this study were to examine the role of transcription and the coincident involvement of type I and type II protein kinase A (PKA) in the resumption of meiosis in murine cumulus-oocyte complexes (COCs) using the transcriptional inhibitors 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) and alpha-amanitin. The first series of experiments was designed to: (i) characterize the role of transcription in gonadotrophin-mediated and spontaneous maturation of murine oocytes; (ii) examine the roles of specific gonadotrophins (FSH versus hCG) and cumulus cells in transcriptionally mediated oocyte maturation; and (iii) determine the reversibility of the transcriptional arrest of meiosis. In the presence of FSH, transcriptional inhibitors arrested germinal vesicle breakdown (GVBD) (DRB: 2 +/- 2% and control: 76 +/- 2%; alpha-amanitin: 4 +/- 4% and control: 70 +/- 4%).

An allele-specific hammerhead ribozyme gene therapy for a porcine model of autosomal dominant retinitis pigmentosa.

Purpose: To develop a hammerhead ribozyme-based gene therapy for a porcine model of autosomal dominant retinitis pigmentosa (ADRP).

Methods: Hammerhead ribozymes were developed and assayed in vitro against RNA targets homologous to the opsin P347S mutants found in a transgenic porcine model and in humans. Both cloned and synthetic RNA oligonucleotide versions of ribozymes and targets were tested under multiple-turnover conditions using oligonucleotide RNA targets.

Transgenic animals as models for human disease.

Transgenic animals, especially mice, have been used quite extensively as models for various human diseases. At first, the level of scientific inquiry was driven by the need to establish the model. In many cases, these models may be considered quite crude because of their limitations.

Early loss of synaptic protein PSD-95 from rod terminals of rhodopsin P347L transgenic porcine retina.

Retinitis pigmentosa (RP), a type of retinal degeneration involving first rod and then slow cone photoreceptor degeneration, can be caused by any of a number of mutations in different genes. In the cases of mutations affecting rod-specific genes such as rhodopsin, it is unclear how the mutations may cause degeneration of cones. We have used the porcine retina, which is rod-dominated and has an abundance of cones, to study the mutation-induced changes in both rod and cone photoreceptors.