At the onset of mammalian neurogenesis, neuroepithelial (NE) cells switch from proliferative to neuron-generating divisions. Understanding the molecular basis of this switch requires the ability to distinguish between these two types of division. Here we show that in the mouse ventricular zone, expression of the mRNA of the antiproliferative gene TIS21 (PC3, BTG2) (i) starts at the onset of neurogenesis, (ii) is confined to a subpopulation of NE cells that increases in correlation with the progression of neurogenesis, and (iii) is not detected in newborn neurons. Expression of the TIS21 mRNA in the NE cells occurs transiently during the cell cycle, i.e., in the G 1 phase. In contrast to the TIS21 mRNA, the TIS21 protein persists through the division of NE cells and is inherited by the neurons, where it remains detectable during neuronal migration and the initial phase of differentiation. Our observations indicate that the TIS21 gene is specifically expressed in those NE cells that, at their next division, will generate postmitotic neurons, but not in proliferating NE cells. Using TIS21 as a marker, we find that the switch from proliferative to neuron-generating divisions is initiated in single NE cells rather than in synchronized neighboring cells.Neuroepithelial (NE) cells are the progenitors of all neurons and macroglial cells of the mammalian central nervous system (CNS). At the onset of CNS neurogenesis, NE cells are thought to switch from symmetric proliferative divisions (two NE daughter cells) to asymmetric neuron-generating divisions (one NE daughter cell, one postmitotic neuron) (1-4). Although several classes of molecules, including growth factors (5) and the products of neurogenic genes (6), have been implicated in this switch, its precise molecular mechanism is unknown. A major problem lies in the fact that during neurogenesis, proliferative and neuron-generating divisions of NE cells coexist (1-3). It is therefore essential to be able to distinguish between proliferating and neuron-generating NE cells. Here we investigate, for the early phase of neurogenesis in the mouse CNS, whether the product of the antiproliferative gene TIS21 (PC3, BTG2) (7-10), that is expressed in the neuroepithelium in correlation with neurogenesis (11), is a specific marker of neuron-generating NE cells. METHODS Morphology.Cryosections. For all combined in situ hybridization (ISH)/immunoperoxidase stainings and for some immunofluorescence, cryosections (5-6 m) were prepared from embryos (either BrdUrd-labeled or unlabeled) fixed overnight at 4°C in 4% paraformaldehyde/4% sucrose in PBS.Polyester Sections. In most double immunofluorescence experiments, polyester sections (6 m) were used. Embryos were fixed overnight at 4°C in 4% paraformaldehyde/0.1% glutaraldehyde in PBS, dehydrated in ethanol, and embedded in polyester wax (BDH) (12).Combined ISH/immunoperoxidase staining on cryosections was performed as described (13). Digoxygenin-labeled sense and antisense TIS21 riboprobes, used at 500 ng RNA per ml of hybridiza...

The cloning syndrome is a continuum with the consequences of abnormal reprogramming manifest throughout gestation, the neo-natal period, and into adulthood in the cloned generation, but it does not appear to be transmitted to subsequent offspring following sexual reproduction. Most in vivo studies on bovine somatic cell cloning have focused on development during pregnancy and the neo-natal period. In this paper, we report on the viability and health of cloned cattle in adulthood. From our studies at AgResearch, we find that between weaning and 4 years of age, the annual mortality rate in cattle cloned from somatic cells is at least 8%. Although the reasons for death are variable and some potentially preventable, the main mortality factor in this period is euthanasia due to musculoskeletal abnormalities. This includes animals with severely contracted flexor tendons and those displaying chronic lameness, particularly in milking cows. In contrast, no deaths beyond weaning have so far been encountered with the offspring of clones where the oldest animals are 3 years of age. In surviving cloned cattle, blood profiles and other indicators of general physiological function such as growth rate, reproduction, rearing of offspring, and milk production are all within the normal phenotypic ranges.

As the demand for cloned embryos and offspring increases, the need arises for the development of nuclear transfer procedures that are improved in both efficiency and ease of operation. Here, we describe a novel zona-free cloning method that doubles the throughput in cloned bovine embryo production over current procedures and generates viable offspring with the same efficiency. Elements of the procedure include zona-free enucleation without a holding pipette, automated fusion of 5-10 oocyte-donor cell pairs and microdrop in vitro culture. Using this system, zona-free embryos were reconstructed from five independent primary cell lines and cultured either singularly (single-IVC) or as aggregates of three (triple-IVC). Blastocysts of transferable quality were obtained at similar rates from zona-free single-IVC, triple-IVC, and control zona-intact embryos (33%, 25%, and 29%, respectively). In a direct comparison, there was no significant difference in development to live calves at term between single-IVC, triple-IVC, and zona-intact embryos derived from the same adult fibroblast line (10%, 13%, and 15%, respectively). This zona-free cloning method could be straightforward for users of conventional cloning procedures to adopt and may prove a simple, fast, and efficient alternative for nuclear cloning of other species as well.

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The precise rotation of suspended cells is one of the many fundamental manipulations used in a wide range of biotechnological applications such as cell injection and enucleation in nuclear transfer (NT) cloning. Noticeably scarce among the existing rotation techniques is the three-dimensional (3D) rotation of cells on a single chip. Here we present an alternating current (ac) induced electric field-based biochip platform, which has an open-top sub-mm square chamber enclosed by four sidewall electrodes and two bottom electrodes, to achieve rotation about the two axes, thus 3D cell rotation. By applying an ac potential to the four sidewall electrodes, an in-plane (yaw) rotating electric field is generated and in-plane rotation is achieved. Similarly, by applying an ac potential to two opposite sidewall electrodes and the two bottom electrodes, an out-of-plane (pitch) rotating electric field is generated and rolling rotation is achieved. As a prompt proof-of-concept, bottom electrodes were constructed with transparent indium tin oxide (ITO) using the standard lift-off process and the sidewall electrodes were constructed using a low-cost micro-milling process and then assembled to form the chip. Through experiments, we demonstrate rotation of bovine oocytes of ~120 μm diameter about two axes, with the capability of controlling the rotation direction and the rate for each axis through control of the ac potential amplitude, frequency, and phase shift, and cell medium conductivity. The maximum observed rotation rate reached nearly 140° s⁻¹, while a consistent rotation rate reached up to 40° s⁻¹. Rotation rate spectra for zona pellucida-intact and zona pellucida-free oocytes were further compared and found to have no effective difference. This simple, transparent, cheap-to-manufacture, and open-top platform allows additional functional modules to be integrated to become a more powerful cell manipulation system.