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|Title:||Development of vectors allowing efficient heterologous-gene expression in stable myeloma-cell transfectants.|
|Presented at:||University of Leicester|
|Abstract:||This thesis describes the development of expression-plasmid vectors for use in the mouse, myeloma cell-line, J558L. Following introduction of the plasmids into J558L using spheroplast fusion, the vectors allow selection for stably-transfected cells. Selective survival depends on expression from a gpt gene in the plasmid, which encodes the enzyme, xanthine-guanine phosphoribosyltransferase (XGPRT). Expression of XGPRT confers, on J558L, a dominant selectable-resistance to mycophenolic acid in the presence of xanthine and hypoxanthine. The resultant stable gpt+-transfectants can then be screened for expression of a protein encoded by a non-selected gene in the expression plasmid. Initially, a systematic study was carried out to compare the effect on XGPRT expression of different expression elements placed upstream of the gpt coding sequence, in single-gene plasmids. Comparative expression levels were estimated by comparing the stable gpt+-transfection frequencies of J558L obtained with each plasmid. This study demonstrated the importance of the IgH-gene enhancer for obtaining high-level expression from a transfected gene. A combination of an IgH enhancer and a promoter element, upstream of the gpt coding-sequence, resulted in the highest levels of expression, but the type of promoter used was of only secondary importance. Identified combinations of effective upstream elements were then incorporated into plasmid vectors, which were constructed for expression from heterologous genes encoding proteins of interest. Stable gpt+- transfectants containing a non-selected gene were only obtained following transfection with plasmids which also contained the gpt gene. This was in contrast to situations in which a non-selected gene was cotransfected with a gpt gene which resided on a different plasmid; here, none of the stable gpt+-transfectants screened had cointegrated the non-selected gene with the gpt gene. Different classes of the two-gene expression plasmids were constructed, which differed in the relative orientation and position of the transcription units. The efficiency of the various plasmids was estimated by measuring the expression levels from a model cDNA, encoding chicken lysozyme. Transfectants were isolated which express and secrete biologically-active chicken lysozyme, at levels greater, in molar terms, than the typical level of secretion of endogenous Ig from myeloma cells. In some cases, the stable gpt+-transfection frequencies obtained with two-gene plasmids were low, compared with the transfection frequencies obtained with plasmids containing only the gpt gene. It was proposed that this was due to transcriptional interference between the two genes on the same plasmid. As this phenomenon might also restrict the expression from the non-selected gene, overcoming this effect was considered important in optimising expression. However, attempts to identify and overcome the phenomenon were inconclusive and, consequently, a rational strategy for overcoming this potential limitation on expression levels was not obtained.|
|Rights:||Copyright © the author. All rights reserved.|
|Appears in Collections:||Theses, Dept. of Biology|
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