Winston T.K. Cheng

Production of Cloned Pigs by Whole-Cell Intracytoplasmic Microinjection

Abstract Cloning by somatic cell nuclear transfer has been successfully achieved by both fusing of a donor cell with and injecting an isolated donor cell nucleus into an enucleated oocyte. However, each of the above methods involves extended manipulation of either the oocytes (fusion) or the donor cells (nucleus isolation). Additionally, cloning efficiency can be reduced by low fusion rate of the cell fusion method, and specialized micromanipulation equipment and exacting nucleus isolation techniques are required for the nucleus injection method. Here we report a whole-cell injection technique for nuclear transfer in pigs and the production of cloned piglets with comparable, if not higher, efficiency than the other two nuclear transfer procedures. First, we tested the feasibility of this technique with three types of frequently used donor cells (cumulus, mural granulosa, and fibroblasts) and obtained the optimal nuclear reprogramming conditions for these cells. We further improved our protocol by avoiding ultraviolet exposure during enucleation and achieved a 37% blastocyst rate. We then conducted whole-cell injection using skin fibroblasts from the ear of a sow transgenic for two genes, the porcine lactoferrin and the human factor IX, and produced four live-born cloned transgenic piglets from three recipients. The present study demonstrated the applicability of producing normal, cloned piglets by the simple and less labor-intensive whole-cell intracytoplasmic injection.

Protective effects of adiponectin against renal ischemia-reperfusion injury via prostacyclin-PPARα-Heme oxygenase-1 signaling pathway

Adiponectin (APN), a circulating adipose‐derived hormone that regulates inflammation and energy metabolism, has beneficial effects on the cardiovascular disorders. Serum APN levels are lower in patients with coronary artery disease and higher in patients with chronic kidney disease. However, the precise role of APN in acute reno‐vascular disease is not clear. Results of the present study show that serum APN concentration decreased after renal ischemia reperfusion (I/R) injury in mice. In addition, I/R‐induced renal dysfunction (elevated serum creatinine and urea levels), inflammation (number of infiltrating neutrophils, myeloperoxidase activity), and apoptotic responses (apoptotic cell number and caspase‐3 activation) were attenuated in APN‐treated compared to control mice. Molecular and biochemical analysis revealed that APN up‐regulates heme oxygenase‐1 (HO‐1) via peroxisome‐proliferator‐activated‐receptor‐α (PPARα) dependent pathway which is mediated through the enhancement of COX‐2 and 6‐keto PGF1α expression. Chromatin immune‐precipitation assay demonstrated that APN increases the binding activity of PPARα to PPRE region of HO‐1 promoter. Furthermore, APN induced HO‐1 expression was only found in wild‐type but not in PPARα gene deleted mice. This provides in vivo evidence that APN mediated HO‐1 expression depends on PPARα regulation. In conclusion, our results provide a novel APN mediated prostacyclin‐PPARα‐HO‐1 signaling pathway in protecting renal I/R injury. J. Cell. Physiol. 227: 239–249, 2012.

Recombinant porcine lactoferrin expressed in the milk of transgenic mice protects neonatal mice from a lethal challenge with enterovirus type 71

The human Enterovirus genus of the piconavirus family causes most of the febrile illnesses that affect children during the summer season in Taiwan. Enterovirus type 71 (EV71) plays a key role in patients with hand-foot-and-mouth disease (HFMD) combined with severe paralysis or encephalitis. It is important to find a method for preventing infection with EV71 since there is no antiviral agent or vaccine for humans. In this study, we developed a transgenic mouse model for demonstrating the protective effects of recombinant lactoferrin (LF) against EV71 infection. Transgenic mice carrying alpha-lactalbumin-porcine lactoferrin (alphaLA-pLF) and BALB/c wild-type mice were subjected to EV71 inoculation. First, we analyzed the expression efficiencies of recombinant pLF (rpLF) in hemizygous and homozygous transgenic mice. Following EV71 inoculation on the 4th day of life, pups ingesting transgenic milk showed the significantly higher survival rate and heavier body weight compared to wild-type mice. RT-PCR analysis for EV71 viral RNA showed that the recombinant pLF had a blocking effect on EV71 infection. Our data suggest that oral intake of pLF-enriched milk exhibited the ability to prevent infection with EV71. The study also provides an animal model for validating the protective effects of pLF.

Gender determination in single bovine blastomeres by polymerase chain reaction amplification of sex-specific polymorphic fragments in the amelogenin gene†

A sensitive technique for the sexing of bovine embryos was developed using polymerase chain reaction (PCR) amplification of the bovine amelogenin (bAML) gene on the X‐ and Y‐chromosomes of Holstein dairy cattle. Cloning and DNA sequencing showed a 45.1% homology between the fifth intron of the bAML‐X and bAML‐Y gene with multiple deletions. A pair of sex‐specific primers was designed to allow amplification of a single fragment of 467‐bp from the X‐chromosome of female cattle and two fragments of 467‐bp and 341‐bp from the X‐ and Y‐chromosomes of male cattle. The primers were successfully applied to bovine sexing from single blastomeres isolated from day‐6 to day‐7 cow embryos by direct cell lysis and PCR. Our protocol of embryo sexing should be applicable to the diagnosis of defective genes in vitro in human embryos and in other domestic or recreational animals. Mol. Reprod. Dev. 54:209–214, 1999.

Temporal and spatial expression of biologically active human factor VIII in the milk of transgenic mice driven by mammary-specific bovine alpha-lactalbumin regulation sequences

Hemophilia A is one of the major inherited bleeding disorders caused by a deficiency or abnormality in coagulation factor VIII (FVIII). Hemophiliacs have been treated with whole plasma or purified FVIII concentrates. The risk of transmitting blood-borne viruses and the cost of highly purified FVIII are the major factors that restrict prophylaxis in hemophilia therapy. One of the challenges created by the biotechnology revolution is the development of methods for the economical production of highly purified proteins in large scales. Recent developments indicate that manipulating milk composition using transgenesis has focused mainly on the mammary gland as a bioreactor to produce pharmaceuticals. In the present study, a hybrid gene containing bovine α-lactalbumin and human FVIII cDNA was constructed for microinjection into the pronuclei of newly fertilized mouse eggs. The αLA-hFVIII hybrid gene was confirmed to be successfully integrated and stably germ-line transmitted in 12 (seven females/five males) lines. Western-blot analysis of milk samples obtained from eight of the transgenic founders and F1 offspring indicated that the recombinant hFVIII was secreted into the milk of the transgenic mice. The concentrations of rFVIII ranged from 7.0 to 50.2 μg/ml, over 35–200-fold higher than that in normal human plasma. Up to 13.4 U/ml of rFVIII was detected in an assay in which rFVIII restored normal clotting activity to FVIII-deficient human plasma.

Caveolin-1 sensitizes rat pituitary adenoma GH3 cells to bromocriptine induced apoptosis

BackgroundProlactinoma is the most frequent pituitary tumor in humans. The dopamine D2 receptor agonist bromocriptine has been widely used clinically to treat human breast tumor and prolactinoma through inhibition of hyperprolactinemia and induction of tumor cell apoptosis, respectively, but the molecular mechanism of bromocriptine induction of pituitary tumor apoptosis remains unclear. Caveolin-1 is a membrane-anchored protein enriched on caveolae, inverted flask-shaped invaginations on plasma membranes where signal transduction molecules are concentrated. Currently, caveolin-1 is thought to be a negative regulator of cellular proliferation and an enhancer of apoptosis by blocking signal transduction between cell surface membrane receptors and intracellular signaling protein cascades. Rat pituitary adenoma GH3 cells, which express endogenous caveolin-1, exhibit increased apoptosis and shrinkage after exposure to bromocriptine. Hence, the GH3 cell line is an ideal model for studying the molecular action of bromocriptine on prolactinoma.ResultsThe expression of endogenous caveolin-1 in GH3 cells was elevated after bromocriptine treatment. Transiently expressed mouse recombinant caveolin-1 induced apoptosis in GH3 cells by enhancing the activity of caspase 8. Significantly, caveolin-1 induction of GH3 cell apoptosis was sensitized by the administration of bromocriptine. Phosphorylation of caveolin-1 at tyrosine 14 was enhanced after bromocriptine treatment, suggesting that bromocriptine-induced phosphorylation of caveolin-1 may contribute to sensitization of apoptosis in GH3 cells exposed to bromocriptine.ConclusionOur results reveal that caveolin-1 increases sensitivity for apoptosis induction in pituitary adenoma GH3 cells and may contribute to tumor shrinkage after clinical bromocriptine treatment.

Molecular Analysis of Cellular Loci Disrupted by Papillomavirus 16 Integration in Cervical Cancer: Frequent Viral Integration in Topologically Destabilized and Transcriptionally Active Chromosomal Regions

To discern the structural features of cellular loci that are disrupted by type 16 human papillomavirus (HPV‐16) integration in cervical cancer, a polymerase chain reaction (PCR)‐based strategy was employed for direct amplification and sequence analysis of four such cellular loci in cancer biopsy samples. One of the HPV‐16‐disrupted loci was found to be the microtubule‐associated protein (MAP‐2) gene and the other three loci were uncharacterized and were designated PID‐1 to ‐3 (for papillomavirus integration‐disrupted). The junctional sequences of the viral integration sites in the four loci analyzed are bracketed by long tracts of homogeneous purine or pyrimidine or alternating purine‐pyrimidine which are known to destabilize the B‐form conformation of the DNA structure. Using a panel of human/hamster hybrid cell DNAs and PCR analysis, the four loci were assigned to chromosomes 2 (MAP‐2), 9 (PID‐1), 1 (PID‐2) and 8 (PID‐3), respectively. These chromosomes carry numerous other previously determined viral integration and chromosomal fragile sites and the myc oncogenes. The PID‐1 locus was further found in Southern analysis to be rearranged and amplified in another cervical cancer biopsy and a cervical carcinoma cell line (CaSki). On Northern analysis, the PID‐1 and ‐3 probes detected a 3.0‐ and a 3.6‐kb transcript, respectively, in normal cervical cells and in cervical cancer cell lines. The findings suggest that HPV‐16 genome integrates frequently into topologically destabilized and transcriptionally active chromosomal sites. It remains to be elucidated whether the MAP‐2 and the PID loci contribute to the pathogenesis of cervical cancer.

Premature Chromosome Condensation Is Not Essential for Nuclear Reprogramming in Bovine Somatic Cell Nuclear Transfer

Abstract Premature chromosome condensation (PCC) was believed to promote nuclear reprogramming and to facilitate cloning by somatic cell nuclear transfer (NT) in mammalian species. However, it is still uncertain whether PCC is necessary for the successful reprogramming of an introduced donor nucleus in cattle. In the present study, fused NT embryos were subjected to immediate activation (IA, simultaneous fusion and activation), delayed activation (DA, activation applied 4 h postfusion), and IA with aged oocytes (IAA, activation at the same oocyte age as group DA). The morphologic changes, such as nuclear swelling, the occurrence of PCC, and microtubule/aster formation, were analyzed in detail by laser-scanning confocal microscopy. When embryos were subjected to IA in both IA and IAA groups, the introduced nucleus gradually became swollen, and a pronuclear-like structure formed within the oocyte, but PCC was not observed. In contrast, delaying embryo activation resulted in 46.5%–91.2% of NT embryos exhibiting PCC. This PCC was observed beginning at 4 h postcell fusion and was shown as one, two, or multiple chromosomal complexes. Subsequently, a diversity of pronuclear-like structures existed in NT embryos, characterized as single, double, and multiple nuclei. In the oocytes exhibiting PCC, the assembled spindle structure was observed to be an interactive mass, closely associated with condensed chromosomes, but no aster had formed. Regardless of whether they were subjected to IA, IAA, or DA treatments, if the oocytes contained pronuclear-like structures, either one or two asters were observed in proximity to the nuclei. A significantly higher rate of development to blastocysts was achieved in embryos that were immediately activated (IA, 59.1%; IAA, 40.7%) than in those for which activation was delayed (14.2%). The development rate was higher in group IA than in group IAA, but it was not significant (P = 0.089). Following embryo transfer, there was no statistically significant difference in the pregnancy rates (Day 70) between two of the groups (group IA, 11.7%, n = 94 vs. group DA, 12.3%, n = 130; P > 0.05) or live term development (group IA, 4.3% vs. group DA, 4.6%; P > 0.05). Our study has demonstrated that the IA of bovine NT embryos results in embryos with increased competence for preimplantational development. Moreover, PCC was shown to be unnecessary for the reprogramming of a transplanted somatic genome in a cattle oocyte.

Isolation and Characterization of Novel Murine Epiphysis Derived Mesenchymal Stem Cells

Background While bone marrow (BM) is a rich source of mesenchymal stem cells (MSCs), previous studies have shown that MSCs derived from mouse BM (BMMSCs) were difficult to manipulate as compared to MSCs derived from other species. The objective of this study was to find an alternative murine MSCs source that could provide sufficient MSCs. Methodology/Principal Findings In this study, we described a novel type of MSCs that migrates directly from the mouse epiphysis in culture. Epiphysis-derived MSCs (EMSCs) could be extensively expanded in plastic adherent culture, and they had a greater ability for clonogenic formation and cell proliferation than BMMSCs. Under specific induction conditions, EMSCs demonstrated multipotency through their ability to differentiate into adipocytes, osteocytes and chondrocytes. Immunophenotypic analysis demonstrated that EMSCs were positive for CD29, CD44, CD73, CD105, CD166, Sca-1 and SSEA-4, while negative for CD11b, CD31, CD34 and CD45. Notably, EMSCs did not express major histocompatibility complex class I (MHC I) or MHC II under our culture system. EMSCs also successfully suppressed the proliferation of splenocytes triggered by concanavalin A (Con A) or allogeneic splenocytes, and decreased the expression of IL-1, IL-6 and TNF-α in Con A-stimulated splenocytes suggesting their anti-inflammatory properties. Moreover, EMSCs enhanced fracture repair, ameliorated necrosis in ischemic skin flap, and improved blood perfusion in hindlimb ischemia in the in vivo experiments. Conclusions/Significances These results indicate that EMSCs, a new type of MSCs established by our simple isolation method, are a preferable alternative for mice MSCs due to their better growth and differentiation potentialities.

Amniotic Fluid Stem Cells Prevent Follicle Atresia and Rescue Fertility of Mice with Premature Ovarian Failure Induced by Chemotherapy

Chemotherapy used to treat cancer may cause irreversible premature ovarian failure (POF). Of late, amniotic fluid stem cells (AFSCs) provide a novel source for regenerative medicine because of their primitive stage, low immunogenicity, and easy accessibility. In this study, we isolated AFSCs from transgenic mice that ubiquitously express enhanced green fluorescence protein (EGFP). These AFSCs exhibited morphologies, immunophenotypes, and mesoderm trilineage differentiation potentials similar to mesenchymal stem cells (MSCs). Further, AFSCs proliferated faster than MSCs and expressed OCT4, a marker for pluripotency. To investigate their potential in recovering fertility in POF model, AFSCs were transplanted into the ovaries of mice with POF six weeks post induction using chemotherapeutic drugs, busulfan and cyclophosphamide. AFSCs could rescue the reproductive ability of mice with POF by preventing follicle atresia and sustaining the healthy follicles. Notably, the transplanted AFSCs did not differentiate into granulosa and germline cells in vivo. After one month, the decreased numbers of transplanted AFSCs accompanied with the reduced beneficial effects indicated that the therapeutic efficacy were directly from AFSCs. These findings demonstrated the therapeutic effects of AFSCs and suggested the promise of AFSCs for treating infertility and POF caused by chemotherapy.