Expressed sequence tags ( EST ) derived from the developing tilapia brain was cloned in our lab. In this study, the Neuro-2a cells was used to investigate the cell cycle ¡Vrelated gene. A stable cell line of Neuro-2a which was transfected with a cell cycel-related gene was established. The flow cytometer was used for the analysis of cell cycle. In a Neruo-2a-CIRP (cold-inducible RNA-binding protein,CIRP) stable cell line, the G2/M phase was prologate in cell cycle.

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Embryonic exposure to the anticonvulsant drug Dilantin has been shown to have detrimental effects on development. Some of the observed effects include growth retardation, craniofacial defects, and even death. As the drug is metabolized, toxic intermediates form, which could be causing the characteristic abnormalities observed in embryos exposed to Dilantin. Culture of preimplantation mouse embryos in the presence of IOµg/mL or 20µglmL Dilantin show a slowing of development inl9.3% and 19.1% ofembryos respectively at Day 3 of culture. The toxic intermediates could be causing alterations in cyclin expression, cell cycle proteins, or the cell cycle timing itself. Previous research determined an in vivo baseline expression for cyclins B 1, E, D, A and cdk2, which was used to compare the expression of these cyclins and cdks between in vivo and Dilantin cultured embryos. Altered patterns in cultured embryos suggested that an alteration in cell cycle timing, therefore, S phase timing was determined in cultured untreated embryos utilizing 5'-Bromo-2-deoxyuridine (BrdU) incorporation and indirect immunofluorescence staining. The results of the experiment showed the second S phase was at 30 hpf, approximately 9 hours later, and the third S phase was at 54 hpf, approximately 3 hours later than previous in vivo literature reports. S phase timing in NaOH vehicle controls did not appear different from untreated controls. Dilantin showed S phase peaks at 24 and 55 hpf. In the Dilantin treated embryos, the nuclear staining intensity for the second S phase did not decrease as rapidly as had been observed in control embryos. Embryos that developed beyond the 2-cell stage demonstrated two distinct S phase peaks at 45 and 54 hpf, while embryos at the 2-cell stage did pass through the second S phase but not the third S phase. These data suggested that the Dilantin could be causing a delay in G2. Future experiments would be necessary to determine if the expression of G2 cyclins are being altered in Dilantin treated embryos. / Department of Biology

Growth is cyclic in nature, periods of activity alternating with periods of comparative rest (PEARL, 1925; WIGGLESWORTH, 1939). Many authors have attempted to construct mathematical expressions for this cyclic growth, in addition to ideal growth curves as an aid in the study of bionomics. Some of these will be dealt with later in this thesis where they prove relevant to the theme. The term “cyclic growth” may be broken down into its constituent parts: (a) it is well known that growth or increase in the bodily dimensions of an animal (or plant) can occur in two main ways, viz. (1) by increase in cell number (cell division); (2) by increase in the size of the cells already present. (Of course, production of cell products may also be considered growth.) The two modes of growth are not mutually exclusive. (b) The definition of “cycle” put forth by KLEITMAN (1949) will be used in this thesis, viz., “A cycle ..... is a repetitive series of events or successive changes of states, thus being either qualitative or quantitative in nature, and its own distinctive feature is one of order of occurrence, rather than duration. Cycles are intrinsic in origin; they may be influenced by inernal and/or external conditions, which affect them quantitatively, but seldom qualitatively.”[...]

In the biopharmaceutical industry, essentially, there are three components that play the main role in producing biopharmaceutical products, the host cell, the expression vector and the bioreactor and/or production environment. To produce the highly valued and desired products, the choice of a suitable host is one of the most important aspects. The host required is not only required to produce the desired product, but also needs to demonstrate robustness in a bioreactor system. Constantly facing challenges in a bioreactor, cells often undergo apoptosis, a well-known limiting factor in biopharmaceutical production, which ultimately leads to low yield of valuable protein(s). We have genetically engineered a CHO-K1 cell line to constitutively express human insulin-like growth factor-1 (IGF-1) and murine polyoma large T-antigen (PyLT-Ag) to generate Super-CHO and CHO-T respectively, two cell lines that can potentially serve different niches in the biopharmaceutical industry. In the first part of the project, we hypothesised that suspension-adapted Super-CHO and CHO-T cells are both resilient cell lines relative to the suspension-adapted CHO-K1 (designated as CHO-XL-99) when facing nutrient depletion, one of the most common problems in a bioreactor. Furthermore, in the second part of this project, the suspension-adapted CHO cell lines were also tested against a cytotoxic heavy metal, cadmium. Without the protection of the metal-resistance element, metallothionein, both Super-CHO and CHO-T cells were also challenged with cadmium to demonstrate their robustness over the parental cell line, CHO-XL-99. In the subsequent study, this project also focussed on the transfection efficiency of each parental and engineered CHO cell lines. Different strategies have been employed in the past in an attempt to improve productivity in the biopharmaceutical industry, from alterations in vector construction, improved culture condition, down to enhanced product recovery. However, the transfer and expression of the gene-of-interest (GOI) has still proven to be the limiting factor for achieving increased specific productivity. In an effort to improve transfection efficiency, strategies including cell cycle synchronisation and various transfection methods to deliver the GOI into the cells have been employed. Thus, the third part of this project has used synchronising agents in conjunction with commercially available lipid- and polymer-based reagents as delivery vehicle for the model protein, EGFP. The combination of cell synchronisation and transfection vehicle on transfection efficiency is studied here, in addition to their individual or collective effect on cell growth, apoptosis and viability. In summary, this project demonstrates the incidence of apoptosis in the cell culture induced by nutrient depletion and heavy metal, and that the use of transfection reagents solely, or in combination with synchronising agents also correlates with the increase of apoptotic indices in the cell culture. The use of the robust cell lines for transfection is an important aspect, and the balance between cell viability and the effort for augmenting transfection efficiency has to be met in order to achieve the maximum biopharmaceutical yields.

Thesis (Ph.D.)--University of Delaware, 2007. / Principal faculty advisor: Melinda K. Duncan, Dept. of Biological Sciences. Includes bibliographical references.

Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2008. / Includes bibliographical references (leaves 149-157). Also available in electronic version.

Thesis (Ph. D.)--University of Texas at Austin, 2001. / Vita. Includes bibliographical references. Available also in a digital version from UMI/Dissertation Abstracts International.

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 83-108).