Optimization of gene transfer in Haliotis midae by means of polyplex mediation

[摘要] ENGLISH ABSTRACT: Haliotis midae is the most important aquaculture species in South Africa, with abalonefarming contributing 80% of the Rand value of the aquaculture industry. Although geneticresearch has benefited the abalone industry, several issues still hinder increases inabalone production. Progress towards an increase in H. midae growth rate by utilizingconventional genetic studies and selective breeding has been relatively slow. Genetransfer has therefore become a plausible option to address this problem. Genes that codefor certain desirable traits, such as increased growth rate, could be incorporated into thegenome of commercial abalone.The current study undertook the optimization of a chemically-mediated gene transfertechnique using Polyethylenimine (PEI) as transfection reagent and fluorescent proteins asreporter genes. Before gene transfer could be undertaken, several complementary studiesalso needed to be undertaken due to the novel nature of the study. The auto fluorescenceof H. midae, the suitability of several H. midae tissues as targets for gene transfer and thecytotoxic effect of transfection reagents and selection antibiotics were assessed beforegene transfer optimization could be attempted. Also, genes linked to an increase in growthrate were characterized for differential expression in different abalone age-groups todetermine the suitability of these genes for incorporation into a homologous gene constructin future transfection studies.The auto fluorescence of ova, embryos and larvae were found to be comparable to that ofthe fluorescent reporter genes, EGFP and DsRed. A PCR-based transfection validationmethod was therefore employed to confirm the presence of internalized transgenes. It wasestablished that sperm, ova, larvae and haemocyte cell culture were the most suitabletarget tissues for transfection. The transfection reagents, a 25kDa PEI and ExGen 500,were not cytotoxic to sperm, embryos and haemocyte cell cultures. The minimum lethalconcentration of the selection antibiotics, neomycin and zeocin, was determined for larvaeand haemocytes. After transfection treatment of sperm and fertilization of untreated ova,the presence of internalized transgenes could be verified for larvae. The presence ofinternalized transgenes could not be detected after transfection treatment of ova andlarvae. Fluorescent flow cytometry and microscopy analysis of haemocytes could notdetect the expression of the fluorescent reporter genes. Expression of two of the growth relatedgenes was found to differ between age-groups. The perlustrin gene was upivregulated in older animals, while the insulin related peptide receptor gene was down regulatedin older animals. The third gene, a thrombospondin-1 precursor was stablyexpressed in all age-groups.This study represents the first report of transfection studies carried out on H. midae. Futurestudies will benefit from the groundwork established in H. midae transfection.

[发布机构] Stellenbosch University