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.