Healthy ageing of cloned sheep

The health of cloned animals generated by somatic-cell nuclear transfer (SCNT) has been of concern since its inception; however, there are no detailed assessments of late-onset, non-communicable diseases. Here we report that SCNT has no obvious detrimental longterm health effects in a cohort of 13 cloned sheep. We perform musculoskeletal assessments, metabolic tests and blood pressure measurements in 13 aged (7–9 years old) cloned sheep, including four derived from the cell line that gave rise to Dolly. We also perform radiological examinations of all main joints, including the knees, the joint most affected by osteoarthritis in Dolly, and compare all health parameters to groups of 5- and 6-year-old sheep, and published reference ranges. Despite their advanced age, these clones are euglycaemic, insulin sensitive and normotensive. Importantly, we observe no clinical signs of degenerative joint disease apart from mild, or in one case moderate, osteoarthritis in some animals. Our study is the first to assess the long-term health outcomes of SCNT in large animals

Tissue engineering, stem cells, cloning, and parthenogenesis: new paradigms for therapy

Patients suffering from diseased and injured organs may be treated with transplanted organs. However, there is a severe shortage of donor organs which is worsening yearly due to the aging population. Scientists in the field of tissue engineering apply the principles of cell transplantation, materials science, and bioengineering to construct biological substitutes that will restore and maintain normal function in diseased and injured tissues. Both therapeutic cloning (nucleus from a donor cell is transferred into an enucleated oocyte), and parthenogenesis (oocyte is activated and stimulated to divide), permit extraction of pluripotent embryonic stem cells, and offer a potentially limitless source of cells for tissue engineering applications. The stem cell field is also advancing rapidly, opening new options for therapy. The present article reviews recent progress in tissue engineering and describes applications of these new technologies that may offer novel therapies for patients with end-stage organ failure

Compounds used to produce cloned animals are genotoxic and mutagenic in mammalian assays in vitro and in vivo

The compounds 6-dimethylaminopurine and cycloheximide promote the successful production of cloned mammals and have been used in the development of embryos produced by somatic cell nuclear transfer. This study investigated the effects of 6-dimethylaminopurine and cycloheximide in vitro, using the thiazolyl blue tetrazolium bromide colorimetric assay to assess cytotoxicity, the trypan blue exclusion assay to assess cell viability, the comet assay to assess genotoxicity, and the micronucleus test with cytokinesis block to test mutagenicity. In addition, the comet assay and the micronucleus test were also performed on peripheral blood cells of 54 male Swiss mice, 35 g each, to assess the effects of the compounds in vivo. The results indicated that both 6-dimethylaminopurine and cycloheximide, at the concentrations and doses tested, were cytotoxic in vitro and genotoxic and mutagenic in vitro and in vivo, altered the nuclear division index in vitro, but did not diminish cell viability in vitro. Considering that alterations in DNA play important roles in mutagenesis, carcinogenesis, and morphofunctional teratogenesis and reduce embryonic viability, this study indicated that 6-dimethylaminopurine and cycloheximide utilized in the process of mammalian cloning may be responsible for the low embryo viability commonly seen in nuclear transfer after implantation in utero.Universidade Estadual Paulista Instituto de Biociências de Rio Claro Programa de Pós-Graduação em Biologia Celular e MolecularUniversidade Federal de Mato Grosso do Sul Núcleo de Hospital Universitário Terapia Celular e Genética ToxicológicaUniversidade Federal de Mato Grosso do Sul Faculdade de Medicina ?Dr. Hélio Mandetta? Programa de Pós-Graduação em Saúde em Desenvolvimento na Região Centro-OesteUniversidade Federal de Mato Grosso do Sul Centro de Ciências Biológicas e da Saúde Programa de Mestrado em FarmáciaUniversidade Estadual de Londrina Departamento de Biologia GeralUniversidade Federal de Mato Grosso do Sul Programa de Doutorado em Biotecnologia e Biodiversidade - Rede Pró Centro-OesteUniversidade Estadual Paulista Faculdade de Medicina de Botucatu Programa de Pós-Graduação em PatologiaUniversidade Estadual Paulista Instituto de Biociências de Rio Claro Programa de Pós-Graduação em Biologia Celular e MolecularUniversidade Estadual Paulista Faculdade de Medicina de Botucatu Programa de Pós-Graduação em Patologi

Parthenogenic Blastocysts Derived from Cumulus-Free In Vitro Matured Human Oocytes

Approximately 20% of oocytes are classified as immature and discarded following intracytoplasmic sperm injection (ICSI) procedures. These oocytes are obtained from gonadotropin-stimulated patients, and are routinely removed from the cumulus cells which normally would mature the oocytes. Given the ready access to these human oocytes, they represent a potential resource for both clinical and basic science application. However culture conditions for the maturation of cumulus-free oocytes have not been optimized. We aimed to improve maturation conditions for cumulus-free oocytes via culture with ovarian paracrine/autocrine factors identified by single cell analysis..Human cumulus-free oocytes from hormone-stimulated cycles are capable of developing to blastocysts when cultured with ovarian factor supplementation. Our improved IVM culture conditions may be used for obtaining mature oocytes for clinical purposes and/or for derivation of embryonic stem cells following parthenogenesis or nuclear transfer

Chemotherapy-Induced Late Transgenerational Effects in Mice

To our knowledge, there is no report on long-term reproductive and developmental side effects in the offspring of mothers treated with a widely used chemotherapeutic drug such as doxorubicin (DXR), and neither is there information on transmission of any detrimental effects to several filial generations. Therefore, the purpose of the present paper was to examine the long-term effects of a single intraperitoneal injection of DXR on the reproductive and behavioral performance of adult female mice and their progeny. C57BL/6 female mice (generation zero; G0) were treated with either a single intraperitoneal injection of DXR (G0-DXR) or saline (G0-CON). Data were collected on multiple reproductive parameters and behavioral analysis for anxiety, despair and depression. In addition, the reproductive capacity and health of the subsequent six generations were evaluated. G0-DXR females developed despair-like behaviors; delivery complications; decreased primordial follicle pool; and early lost of reproductive capacity. Surprisingly, the DXR-induced effects in oocytes were transmitted transgenerationally; the most striking effects being observed in G4 and G6, constituting: increased rates of neonatal death; physical malformations; chromosomal abnormalities (particularly deletions on chromosome 10); and death of mothers due to delivery complications. None of these effects were seen in control females of the same generations. Long-term effects of DXR in female mice and their offspring can be attributed to genetic alterations or cell-killing events in oocytes or, presumably, to toxicosis in non-ovarian tissues. Results from the rodent model emphasize the need for retrospective and long-term prospective studies of survivors of cancer treatment and their offspring

Development of Transgenic Cloned Pig Models of Skin Inflammation by DNA Transposon-Directed Ectopic Expression of Human β1 and α2 Integrin

Integrins constitute a superfamily of transmembrane signaling receptors that play pivotal roles in cutaneous homeostasis by modulating cell growth and differentiation as well as inflammatory responses in the skin. Subrabasal expression of integrins α2 and/or β1 entails hyperproliferation and aberrant differentiation of keratinocytes and leads to dermal and epidermal influx of activated T-cells. The anatomical and physiological similarities between porcine and human skin make the pig a suitable model for human skin diseases. In efforts to generate a porcine model of cutaneous inflammation, we employed the Sleeping Beauty DNA transposon system for production of transgenic cloned Göttingen minipigs expressing human β1 or α2 integrin under the control of a promoter specific for subrabasal keratinocytes. Using pools of transgenic donor fibroblasts, cloning by somatic cell nuclear transfer was utilized to produce reconstructed embryos that were subsequently transferred to surrogate sows. The resulting pigs were all transgenic and harbored from one to six transgene integrants. Molecular analyses on skin biopsies and cultured keratinocytes showed ectopic expression of the human integrins and localization within the keratinocyte plasma membrane. Markers of perturbed skin homeostasis, including activation of the MAPK pathway, increased expression of the pro-inflammatory cytokine IL-1α, and enhanced expression of the transcription factor c-Fos, were identified in keratinocytes from β1 and α2 integrin-transgenic minipigs, suggesting the induction of a chronic inflammatory phenotype in the skin. Notably, cellular dysregulation obtained by overexpression of either β1 or α2 integrin occurred through different cellular signaling pathways. Our findings mark the creation of the first cloned pig models with molecular markers of skin inflammation. Despite the absence of an overt psoriatic phenotype, these animals may possess increased susceptibility to severe skin damage-induced inflammation and should be of great potential in studies aiming at the development and refinement of topical therapies for cutaneous inflammation including psoriasis