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Publication type: Diploma Thesis

Year: 2013

Author(s): Maier, Judith

Title: Quantifizierung des Konstrukts im Kerngenom r28m transgener Rinder und Korrelation der Kopienzahl mit der Proteinexpression.

Other title: Quantification of the construct in the genome of r28m transgene cattle and correlation of the copy number wiht the protein expression

Source: Diplomarbeit, Vet. Med. Univ. Wien, pp. 31.


Advisor(s):

Brem Gottfried

Reviewer(s):
Steinborn Ralf

Vetmed Research Units:
Institute of Animal Breeding and Genetics


Graduation date: 29.07.13


Abstract:
r28m is an antibody directed to a melanoma-associated proteoglycan. It is assumed that r28m stimulates the CD28 molecule, which is also expressed on NK cells and so leads to the lysis of the tumor cells. Transgenic animals gained increased importance during the last years to express a protein of our choice. In contrast to conventional expression systems, like bacteria cultures or yeast, antibodies are here produced in sufficient number and quality. The aim of this research was to quantify the construct in the genome of r28m transgenic cattle and to compare the protein expression with the number of copies in the genome. The analysis of the blood samples of r28m positive transgenic animals was conducted employing qPCR.We found that all transgenic animals were hemizygote and had an average of 18 copies in their genome. Four animals inherited the construct homozygotic, their copy number of constructs in the genome was twice the number of the others. However, all four homozygotic animals died within one year of yet unexplained causes. The protein expression data was provided by the „Christian Doppler Labor für Innovative Immuntherapie“ of the VMU Vienna. Since only hemizygotic animals with an identical r28m copy number were available to us, we could not show any correlation concerning the protein expression. However, this is nevertheless an important finding as it is now clear that the apparent changes in protein expression have to have other reasons. In further studies, the genome of r28m transgenes could be sequenced to identify the exact integration site of the construct in the genome. That way we could perhaps find out whether or not the death of the homozygotic animals is connected to the integrated construct. Furthermore, our results could be checked again with dPCR, a method, which detects even smaller differences in the copy number than qPCR.


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