Thesis (MSc (Genetics))--Stellenbosch University, 2010. / ENGLISH ABSTRACT: Haliotis midae is the most important aquaculture species in South Africa, with abalone
farming contributing 80% of the Rand value of the aquaculture industry. Although genetic
research has benefited the abalone industry, several issues still hinder increases in
abalone production. Progress towards an increase in H. midae growth rate by utilizing
conventional genetic studies and selective breeding has been relatively slow. Gene
transfer has therefore become a plausible option to address this problem. Genes that code
for certain desirable traits, such as increased growth rate, could be incorporated into the
genome of commercial abalone.
The current study undertook the optimization of a chemically-mediated gene transfer
technique using Polyethylenimine (PEI) as transfection reagent and fluorescent proteins as
reporter genes. Before gene transfer could be undertaken, several complementary studies
also needed to be undertaken due to the novel nature of the study. The auto fluorescence
of H. midae, the suitability of several H. midae tissues as targets for gene transfer and the
cytotoxic effect of transfection reagents and selection antibiotics were assessed before
gene transfer optimization could be attempted. Also, genes linked to an increase in growth
rate were characterized for differential expression in different abalone age-groups to
determine the suitability of these genes for incorporation into a homologous gene construct
in future transfection studies.
The auto fluorescence of ova, embryos and larvae were found to be comparable to that of
the fluorescent reporter genes, EGFP and DsRed. A PCR-based transfection validation
method was therefore employed to confirm the presence of internalized transgenes. It was
established that sperm, ova, larvae and haemocyte cell culture were the most suitable
target tissues for transfection. The transfection reagents, a 25kDa PEI and ExGen 500,
were not cytotoxic to sperm, embryos and haemocyte cell cultures. The minimum lethal
concentration of the selection antibiotics, neomycin and zeocin, was determined for larvae
and haemocytes. After transfection treatment of sperm and fertilization of untreated ova,
the presence of internalized transgenes could be verified for larvae. The presence of
internalized transgenes could not be detected after transfection treatment of ova and
larvae. Fluorescent flow cytometry and microscopy analysis of haemocytes could not
detect the expression of the fluorescent reporter genes. Expression of two of the growth related
genes was found to differ between age-groups. The perlustrin gene was upiv
regulated in older animals, while the insulin related peptide receptor gene was down regulated
in older animals. The third gene, a thrombospondin-1 precursor was stably
expressed in all age-groups.
This study represents the first report of transfection studies carried out on H. midae. Future
studies will benefit from the groundwork established in H. midae transfection. / AFRIKAANSE OPSOMMING: Haliotis midae is die belangrikste akwakultuur spesie in Suid-Afrika met perlemoen
boerdery wat 80% van die Rand waarde van die akwakultuur industrie bydrae. Alhoewel
genetiese studies die perlemoen industrie ‘n hupstoot gegee het, is daar steeds sekere
struikelblokke wat verdere toename in produksie verhoed. Vooruitgang ten opsigte van ‘n
toename in H. midae se groei tempo deur gebruik te maak van konvensionele genetiese
studies en selektiewe teling was tot dusver relatief stadig. Genetiese transformasie het
daarom ‘n wesenlike alternatief geword wat moontlik hierdie probleem kan oplos. Gene
wat kodeer vir sekere eienskappe, soos ‘n toename in groeitempo, kan in die genoom van
kommersiële perlemoen inkorporeer word.
Die huidige studie het onderneem om ‘n chemies-gemedieerde genetiese transfeksie
tegniek te optimiseer en van Polyethylenimine (PEI) as transfeksie reagens en
fluoresserende proteine as verklikkers gebruik te maak. As gevolg van die
oorspronklikheid van die studie moes verskeie bykomende ondersoeke ook aangepak
word voordat genetiese transfeksie uitgevoer kon word. Die outofluoressensie van H.
midae, die geskiktheid van verskeie H. midae teiken weefsels en die sitotoksiese effek van
die transfeksie reagense en seleksie antibiotika is ondersoek voordat transfeksie uitgevoer
is. Gene gekoppel aan ‘n toename in groeitempo is ook gekarakteriseer vir verskille in
uitdrukking in verskillende perlemoen ouderdoms-groepe om te bepaal of hierdie gene
moontlik in ‘n homoloë geen konstruk ingesluit kan word vir toekomstige transfeksie
studies.
Dit is gevind dat die outofluoressensie van ova, embrios en larwes vergelykbaar is met
die fluoressensie van die verklikker proteïene, EGFP en DsRed. ‘n PKR-baseerde metode
om die internalisering van die transgeen te kontroleer is daarom gebruik. Dit is vasgestel
dat sperm, ova, larwes en haemosiete die mees geskikte teiken vir transfeksie sou wees.
Die transfeksie reagense, ‘n 25kDa PEI en Exgen 500, is nie sitotoksies vir sperm,
embrios of haemosiete nie. Die minimum dodelike konsentrasie van die seleksie
antibiotika, neomycin en zeocin, is bepaal. Na transfeksie behandeling van sperm en
bevrugting van onbehandelde ova, kon die teenwoordigheid van internaliseerde transgene
bevestig word vir larwes. Die teenwoordigheid van internaliseerde transgene kon nie
bevestig word na transfeksie behandeling van ova en larwes nie. Fluoressente vloei
sitometrie en mikroskopiese analise kon nie die uitdrukking van die fluoressente verklikker
gene bevestig in haemosiete nie. Die uitdrukking van twee van die gene gekoppel aan
groei het verskil tussen ouderdomsgroepe. Die perlustrin geen is meer uitgedruk in ouer
diere terwyl die insulien geassosieerde peptied reseptor geen minder uitgedruk is in ouer
diere. Die thrombospondin-1 voorloper geen is stabiel uitgedruk in al die ouderdomsgroepe.
Hierdie studie verteenwoordig die eerste verslag van transfeksie studies uitgevoer op H.
midae. Toekomstige studies sal baat vind by die grondslag wat deur hierdie projek gelê is.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/5478 |
| Date | 12 1900 |
| Creators | Sandenbergh, Lise |
| Contributors | Roodt-Wilding, R., Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | xxi, 139 p. : ill. |
| Rights | Stellenbosch University |
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