Expression of phospholipase c zeta (PLCzeta) in a mammalian cell line

Phillips, Sally Victoria 2009. Expression of phospholipase c zeta (PLCzeta) in a mammalian cell line. PhD Thesis, Cardiff University.

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Abstract

Phospholipase C zeta (PLCzeta) is a sperm specific isoform of phospholipase C. It has been shown to produce a long lasting series of calcium oscillations and triggers the activation of development when introduced into mammalian eggs. It is not known how PLCzeta is regulated or if its effects are specific to eggs. Here Chinese Hamster Ovary (CHO) cells were transfected with cDNA encoding PLCzeta tagged with enhanced yellow fluorescent protein (eYFP) or luciferase (LUC). Comparisons were made between these cells and cells transfected with the catalytically inactive PLCzeta, the corresponding reporter gene, or nontransfected cells. PLCzeta exhibited variable levels of nuclear localisation in a manner that depended upon time after transfection. Analysis of resting intracellular calcium levels in transfected CHO cells produced no evidence that PLCzeta expression has a significant effect upon calcium homeostasis. The calcium response to ATP receptor stimulation also remained unchanged after PLCzeta expression. A lack of any clear effect on cell viability enabled the generation of a stably transfected PLCzeta cell line. Individual cells were estimated to be expressing PLCzeta within and above the range required to initiate calcium transients in eggs, and are therefore considered to be expressing at levels comparable with that of sperm. Despite the lack of effect on calcium in CHO cells, the injection of either cytosolic extracts, or whole cells, from the PLCzeta transfected cell line were able to cause calcium oscillations in mouse eggs. Such an effect was not seen with control CHO cells. These data suggest that PLCzeta is inactive when expressed in CHO cells and yet active when subsequently introduced into an egg. The results imply that the enzymatic activity of PLCzeta may be reversibly inhibited in somatic cells, or else specifically stimulated by factors in the egg cytoplasm.

Item Type:	Thesis (PhD)
Status:	Unpublished
Schools:	Medicine
ISBN:	9781303191138
Date of First Compliant Deposit:	30 March 2016
Last Modified:	03 Oct 2014 14:53
URI:	https://orca.cardiff.ac.uk/id/eprint/55886

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