We have shown previously that transgene expression can be suppressed in hematopoietic cells using vectors that are responsive to microRNA (miRNA) regulation. Here we investigate the potential of this approach for more sophisticated control of transgene expression. Analysis of the relationship between miRNA expression levels and target mRNA suppression suggested that suppression depends on a threshold miRNA concentration. Using this information, we generated vectors that rapidly adjust transgene expression in response to changes in miRNA expression. These vectors sharply segregated transgene expression between closely related states of therapeutically relevant cells, including dendritic cells, hematopoietic and embryonic stem cells, and their progeny, allowing positive/negative selection according to the cells' differentiation state. Moreover, two miRNA target sites were combined to restrict transgene expression to a specific cell type in the liver. Notably, the vectors did not detectably perturb endogenous miRNA expression or regulation of natural targets. The properties of miRNA-regulated vectors should allow for safer and more effective therapeutic applications.

The large offspring syndrome (LOS) is observed in bovine and ovine offspring following transfer of in vitro-produced (IVP) or cloned embryos and is characterized by a multitude of pathologic changes, of which extended gestation length and increased birthweight are predominant features. In the present study, we used bovine blastocysts to analyze cellular parameters, i.e., the number of cells in Day 7 blastocysts and the size of Day 12 elongating blastocysts, and molecular parameters, i.e., the relative abundance of developmentally important genes: glucose transporter (Glut) 1, Glut-2, Glut-3, Glut-4, heat shock protein (Hsp) 70.1, Cu/Zn-superoxide dismutase (SOD), histone H4.1, basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF) I receptor (R), and IGFII-R. Some blastocysts were produced by in vitro maturation and fertilization followed by in vitro culture in synthetic oviduct fluid medium supplemented with BSA or human serum or by in vivo culture in the sheep oviduct. Other blastocysts were derived in vivo from the uterine horns of superovulated donors. The findings made in the early embryos were related to a representative number of calves obtained from each production system and from artificial insemination (AI). In vitro culture of bovine embryos in the presence of high concentrations of serum or BSA significantly increased the number of cells in Day 7 blastocysts, the size of blastocysts on Day 12, and the relative abundance of the transcripts for Hsp70.1, Cu/Zn-SOD, Glut-3, Glut-4, bFGF, and IGFI-R when compared with embryos from the in vivo production groups. Birthweights of calves derived from IVP embryos were significantly higher than those of calves derived from sheep oviduct culture, superovulation, or AI. The results support the hypothesis that persistence of early deviations in development is causally involved in the incidence of LOS, in particular in increased birthweights. The cellular and molecular parameters analyzed in this study can be considered early markers of LOS in cattle.

Several animal species, including sheep, mice, cattle, goats, rabbits, cats, pigs and, more recently, mules have been reproduced by somatic cell cloning, with the offspring being a genetic copy of the animal donor of the nuclear material used for transfer into an enucleated oocyte. Here we use this technology to clone an adult horse and show that it is possible to establish a viable, full-term pregnancy in which the surrogate mother is also the nuclear donor. The cloned offspring is therefore genetically identical to the mare who carried it, challenging the idea that maternal immunological recognition of fetal antigens influences the well-being of the fetus and the outcome of the pregnancy.

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Background: Glutaraldehyde fixation does not guarantee complete tissue biocompatibility in current clinical bioprosthetic heart valves (BHVs). Particularly, circulating anti-aGal human antibodies increase significantly from just 10 days after a BHV implantation. The inactivation of such epitope should be mandatory to meet the requirements for a perspectively safe clinical application; nevertheless, its quantitative assessment in commercially available BHVs has never been carried out. Methods: In this investigation, seven different models of BHVs were tested. The number of epitopes was determined with reference to a standard aGal source by an ELISA test. The presence of xenoantigen was subsequently confirmed by immunofluorescence analysis. Porcine tissue, knockout for the aGal epitopes, was used as negative control. Results: Epic TM valve was the only model among those tested, in which the aGal antigen appeared to be completely shielded. Composite Trifecta TM valve exhibited conflicting results: cusps of bovine pericardial tissue were devoid of reactive aGal epitopes, while the stent cover strip of porcine pericardium still maintained 30% of active antigens originally present in native tissue. All other tested BHVs express an aGal amount not significantly different from that exhibited by porcine Mosaic â valve (5.2 AE 0.6 9 10 10 each 10 mg of tissue). Conclusions: For the first time, the quantitative evaluation of the aGal epitope in heart valve bioprostheses, already in clinical practice for about 40 yrs, was finally determined. Such quantification might provide indications of biocompatibility relevant for the selection of bioprosthetic devices and an increase in the confidence of the patient. It might become a major quality control tool in the production and redirection of future investigation in the quest for aGal-free long-lasting substitutes.