Clinical application of amniotic membrane as a biologic dressing in oral cavity and pharyngeal defects after tumor resection.

Background: Oropharyngeal malignancies represent management challenges for the head and neck surgeons. Tumor resection and reconstruction with graft is the standard treatment. Split-thickness skin grafts are routinely used to cover the mucosal defects arising from resections.

Goat embryonic stem-like cell derivation and characterization.

Embryonic stem (ES) cells are derived from the inner cell masses of preimplantation embryos. ES cells are pluripotent cells with the capacity for long-term propagation and broad differentiation plasticity. These cells have an exceptional functional feature in that they can differentiate into all tissues and organs, including germ cells.

Establishment of goat embryonic stem cells from in vivo produced blastocyst-stage embryos.

Embryonic stem (ES) cells with the capacity for germ line transmission have only been verified in mouse and rat. Methods for derivation, propagation, and differentiation of ES cells from domestic animals have not been fully established. Here, we describe derivation of ES cells from goat embryos.

Production of recombinant albumin by a herd of cloned transgenic cattle.

Purified plasma derived human albumin has been available as a therapeutic product since World War II. However, cost effective recombinant production of albumin has been challenging due to the amount needed and the complex folding pattern of the protein. In an effort to provide an abundant source of recombinant albumin, a herd of transgenic cows expressing high levels of rhA in their milk was generated.

Laparoscopic ovum pick-up followed by in vitro embryo production for the reproductive rescue of aged goats of high genetic value.

The efficiency of laparoscopic ovum pick-up (LOPU) followed by in vitro embryo production (IVEP) in the propagation of aged goats with poor reproductive performance was evaluated in the present study. Follicular development was stimulated in donor goats with 80 mg follicle-stimulating hormone and 300 IU equine chorionic gonadotrophin administered 36 h before LOPU. In addition, goats were heat synchronised with intravaginal sponges containing 60 mg medroxyprogesterone acetate for 10 days and a luteolytic injection of 125 microg cloprostenol 36 h before sponge removal and LOPU.

Depletion of endogenous germ cells in male pigs and goats in preparation for germ cell transplantation.

The efficiency of germ cell transplantation, the procedure of transferring germ cells from a donor male into the testes of recipient males, can be greatly increased by reduction of endogenous germ cells in recipient animals. To develop effective methods for suppression of endogenous spermatogenesis in potential pig and goat recipients, we either administered busulfan to pregnant sows or irradiated the testes of immature goats. Piglets from sows treated twice with busulfan (7.

Metabolism, protein content, and in vitro embryonic development of goat cumulus-oocyte complexes matured with physiological concentrations of glucose and L-lactate.

No information is available concerning how the maturation environment controls the metabolism of goat oocytes. The objectives of this experiment were to: (1) Determine the concentrations of glucose, lactate, and pyruvate in caprine follicular fluid; and (2) Investigate the effects of physiological concentrations of glucose and lactate in the in vitro maturation (IVM) medium on the metabolism (glycolysis and pyruvate oxidation), protein content, and developmental competence of caprine oocytes and cumulus-oocyte complexes (COCs). Abattoir-derived COCs were matured for 18-20 hr in a defined, SOF-based medium containing 0.

Health and reproductive profiles of malaria antigen-producing transgenic goats derived by somatic cell nuclear transfer.

Nuclear transfer (NT) using transfected primary cells is an efficient approach for the generation of transgenic goats. However, reprogramming abnormalities associated with this process might result in compromised animals. We examined the health, reproductive performance, and milk production of four transgenic does derived from somatic cell NT.

Effect of serum concentration, method of trypsinization and fusion/activation utilizing transfected fetal cells to generate transgenic dairy goats by somatic cell nuclear transfer.

This work was performed within a commercial nuclear transfer program to investigate different methods for synchronizing donor cell cycle stage, for harvesting donor cells, and for fusion and activation of reconstructed caprine embryos. Primary fetal cells isolated from day 35 to day 40 fetuses were co-transfected with DNA fragments encoding both the heavy and light immunoglobulin chains of three different monoclonal antibodies and neomycin resistance. Four neomycin resistant cell lines for each antibody were selected, expanded, and aliquots were both cryopreserved for later use as karyoplast donors or used for further genetic characterization.

Viable transgenic goats derived from skin cells.

The current study was undertaken to evaluate the possibility of expanding transgenic goat herds by means of somatic cell nuclear transfer (NT) using transgenic goat cells as nucleus donors. Skin cells from adult, transgenic goats were first synchronized at quiescent stage (G0) by serum starvation and then induced to exit G0 and proceed into G1. Oocytes collected from superovulated donors were enucleated, karyoplast-cytoplast couplets were constructed, and then fused and activated simultaneously by a single electrical pulse.

Effect of macromolecule supplementation during in vitro maturation of goat oocytes on developmental potential.

In vitro maturation (IVM) of goat oocytes with serum-supplemented media results in oocytes with reduced developmental potential. The objective of this study was to develop a defined medium for IVM of goat oocytes that better supports subsequent embryonic development. Cumulus oocyte complexes (COC) were matured for 18-20 hr in: Experiment (1), tissue culture medium 199 (TCM199) with 10% (v/v) goat serum or modified synthetic oviduct fluid maturation medium (mSOFmat) with 2.

Synchronization of goat fibroblast cells at quiescent stage and determination of their transition from G0 to G1 by detection of cyclin D1 mRNA.

A number of studies have reported that donor cells consisting of serum starved cells, which are assumed to be at quiescence (G0), or non-starved confluent cells or mitotic cells obtained by shake-off, both of which are assumed to be at G1 phase, give better results in nuclear transfer (NT) than cells at other phases of the cell cycle. Whether G0 or G1 cells function better as donor cells is yet to be determined by detailed studies. The aims of this study were to analyze the cell cycle of goat transfected fibroblasts and determine the timing of transition from G0 to G1 by detecting G1-specific marker, cyclin D1 mRNA.

Fertility and germline transmission of donor haplotype following germ cell transplantation in immunocompetent goats.

Transplantation of spermatogonial stem cells into syngeneic or immunosuppressed recipient mice or rats can result in donor-derived spermatogenesis and fertility. Recently, this approach has been employed to introduce a transgene into the male germline. Germ-cell transplantation in species other than laboratory rodents, if successful, holds great promise as an alternative to the inefficient methods currently available to generate transgenic farm animals that can produce therapeutic proteins in their milk or provide organs for transplantation to humans.

Germ cell transplantation in goats.

Transplantation of spermatogonial stem cells provides a unique approach for the study of spermatogenesis and manipulation of the male germ line. This technique may also offer an alternative to the currently inefficient methods of producing transgenic domestic animals. We have recently established the technique of spermatogonial transplantation, originally developed in laboratory rodents, in pigs, and this study was aimed to extend the technique to the goat.

Cloned transgenic offspring resulting from somatic cell nuclear transfer in the goat: oocytes derived from both follicle-stimulating hormone-stimulated and nonstimulated abattoir-derived ovaries.

The use of nuclear transfer (NT) techniques to create transgenic offspring capable of producing valuable proteins may have a major impact on the pharmaceutical market. Our objective was to compare the in vivo developmental potential of NT embryos produced from the fusion of transgenic donor cells with cytoplasts prepared from either FSH-stimulated ovaries or nonstimulated abattoir-derived ovaries. Donor cells were prepared from a transgenic fetus carrying the gene for human antithrombin III as a marker and used within four to eight subpassages.

Transgenic production from in vivo-derived embryos: effect on calf birth weight and sex ratio.

We examined transgenic-cattle production by DNA microinjection into 1-, 2-, and 4-cell embryos, analyzing the impact on calf size and subsequent viability. Embryos were either collected at an abattoir by flushing oviducts from superovulated and artificially inseminated cows (in vivo-derived) or obtained by in vitro maturation and in vitro fertilization of oocytes aspirated from excised ovaries (in vitro-derived). A human serum albumin (hSA) milk-expression DNA construct was microinjected, either in one of the visible pronuclei of in vitro- and in vivo-derived 1-cell embryos or in the nuclei of two blastomeres of 2- and 4-cell in vivo-derived embryos.

Development of goat embryos after in vitro fertilization and parthenogenetic activation by different methods.

Effective activation protocols that can be used during nuclear transfer investigations in goats need to be developed. We compared the development of IVF goat embryos with those of nonfertilized parthogenetically developing oocytes activated by treatment with either ionomycin or ethanol, both followed by immediate exposure to 6-diethylaminopurine (6-DMAP). Cumulus oocyte complexes (COCs) recovered from abattoir goat ovaries were either matured in a conventional laboratory incubator or placed in pre-equilibrated maturation medium and shipped overnight in a battery-operated dry incubator to another laboratory.

Production of goats by somatic cell nuclear transfer.

In this study, we demonstrate the production of transgenic goats by nuclear transfer of fetal somatic cells. Donor karyoplasts were obtained from a primary fetal somatic cell line derived from a 40-day transgenic female fetus produced by artificial insemination of a nontransgenic adult female with semen from a transgenic male. Live offspring were produced with two nuclear transfer procedures.

Isolation of pluripotent stem cells from cultured porcine primordial germ cells.

Embryonic germ (EG) cells are undifferentiated stem cells isolated from cultured primordial germ cells (PGC). To date, EG cells have been isolated only in the mouse. Murine EG cells share several characteristics with embryonic stem (ES) cells, including morphology, pluripotency, and the capacity for germline transmission.

Early transcription of the SRY gene by bovine preimplantation embryos.

We have examined mRNA expression of two genes located on the Y chromosome, the sex-determining region Y gene (SRY) and the linked zinc finger gene (ZFY), using in vitro fertilized-in vitro cultured bovine embryos. Expression of the SRY gene, implicated in sex determination in mammals, has been reported to occur both for a short time at the sex-determining stage of development around the period of the primitive undifferentiated gonad and in the adult testis. In this study, using a sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) assay, we detected SRY but not ZFY mRNA expression as early as the 4- to 8-cell stage and through to the blastocyst stage in bovine embryos.

Phase transition temperature and chilling sensitivity of bovine oocytes.

A limiting factor for achieving cryopreservation of oocytes is direct chilling injury (DCI), which occurs during cooling. DCI, or cold shock, is defined as an irreversible damage expressed shortly after exposure to low, but not freezing, temperatures. The primary target of DCI is thought to be the plasma membrane.

Relationship between stage of development and sex of bovine IVM-IVF embryos cultured in vitro versus in the sheep oviduct.

We have confirmed more rapid development of male compared with female in vitro-cultured bovine embryos during the first 7 d after in vitro fertilization. The male-to-female sex ratio of expanded blastocysts after 10 d of in vitro culture was 1.37:1.