Claydon, Kerry (2009) Advances in crustacean cell culture. PhD thesis, James Cook University.

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Abstract

In 2006, for the first time in history, more seafood was produced from aquaculture raised animals than wild caught fisheries. However, crustacean aquaculture continues to be plagued by disease, due to the lack of sensitive investigative methods that assist diagnostics and pathogen control. Prevention and control of diseases are an absolute priority for the durability of the crustacean aquaculture industry.

The development of a permanent in vitro model for crustacean species is imperative. The research presented herein attempted to utilise novel modern technologies to develop the first continuous cell line from crustacea. The first investigation involved assessing methodologies that explored the existence of crustacean components in hybridised cell lines, which were developed by combining crustacean cells with immortal fish cells (Chapter 4). The methodologies involved cellular assessment from genomic and protein approaches, along with exploration of viral susceptibility of the cells. Using PCR, no crustacean 18S rRNA genes or haemocyanin genes were found in any of the seven hybrid cell lines. No crustacean proteins were detected, nor did any viral amplification occur when hybrid cells were inoculated with two crustacean viruses, indicating that these hybrid cell populations were not suitable for crustacean virological studies (Chapter 5).

The second investigation involved optimisation of in vitro methods using the Australian freshwater crayfish, Cherax quadricarinatus (Chapters 6 and 7). The approach included scientifically optimising the culture medium by comparing cell proliferation with three different cell culture media and 25 different media supplements. Leibovitz-15 medium, along with supplemental iron, copper, foetal bovine serum, and non-essential amino acids, was found to significantly increase cell proliferation (F = 6.231, df = 1,19, p< 0.05) and also augment the longevity of cells in vitro.

While spontaneous transformation of somatic cells can occur, transgenesis often expedites immortality. Therefore, the third investigation explored the transfection of C. quadricarinatus primary cells (Chapter 8). A lipofection reagent was used to introduce an observable green fluorescent protein vector into the cytoplasm of the cells. Transfection of the cells was then attempted using human oncogenes.

Papillomaviruses are non-enveloped, double-stranded DNA tumour viruses that play a critical role in the formation of human anogenital cancer. Early studies have demonstrated that the human papillomavirus-expressed E6 and E7 proteins function concomitantly to disrupt the p53 and Rb tumour suppressor genes, regulators of the cell-cycle checkpoints at the first gap (G1) phase of the cell cycle. To help C. quadricarinatus cells pass through the G1 phase and enter the DNA synthesis stage of the cell cycle, HPV E6 and E7 genes were transfected into the C. quadricarinatus cells. Successful transfection was demonstrated by the presence of oncogene mRNA by RT-PCR. At day 150, transfected cells remained viable, although cell proliferation was stagnant. It may be that, although transfection of the oncogenes was successful, no proliferation of the C. quadricarinatus cells was evident, due to a lack of telomere maintenance.

Overall, attempts to create a crustacean cell line remain elusive. Although the end product of a permanent cell line has not been forthcoming, this research made successful advancements in crustacean cell culture methodologies and explored new techniques and technologies that may assist eventual immortalisation.

Item ID:	11946
Item Type:	Thesis (PhD)
Keywords:	crustaceans, cell cultures, Australian freshwater crayfishes, crayfish culture, aquaculture fishes, crayfish diseases, infectious diseases, farmed crayfish, immortal cell cultures, cell hybridization, cross species infection
Related URLs:	http://eprints.jcu.edu.au/6405
Copyright Information:	Copyright © 2009 Kerry Claydon
Additional Information:	Publications arising from this thesis are available from the Related URLs field. The publications are: Claydon, Kerry, and Owens, Leigh (2008) Attempts at immortalization of crustacean primary cell cultures using human cancer genes. In Vitro Cellular and Developmental Biology: animal, 44 (10). pp. 451-457. ISSN 1543-706X
Date Deposited:	14 Oct 2010 06:14
FoR Codes:	07 AGRICULTURAL AND VETERINARY SCIENCES > 0704 Fisheries Sciences > 070404 Fish Pests and Diseases @ 50% 07 AGRICULTURAL AND VETERINARY SCIENCES > 0704 Fisheries Sciences > 070401 Aquaculture @ 50%
SEO Codes:	83 ANIMAL PRODUCTION AND ANIMAL PRIMARY PRODUCTS > 8301 Fisheries - Aquaculture > 830101 Aquaculture Crustaceans (excl. Rock Lobster and Prawns) @ 100%
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