Localization of human coagulation factor VIII (hFVIII) in transgenic rabbit by FISH-TSA: identification of transgene copy number and transmission to the next generation.

For chromosomal localization of the hFVIII human transgene in F2 and F3 generation of transgenic rabbits, FISH-TSA was applied. A short cDNA probe (1250 bp) targeted chromosomes 3, 7, 8, 9 and 18 of an F2 male (animal 1-3-8). Two transgenic offspring (F3) revealed signal positions in chromosome 3 and chromosomes 3 and 7, respectively. Sequencing and structure analysis of the rabbit orthologous gene revealed high similarity to its human counterpart. Part of the sequenced cDNA (1310 bp) served as a probe for FISH-TSA analysis. The rabbit gene was localized in the q arm terminus of the X chromosome. This result is in agreement with reciprocal chromosome painting between the rabbit and the human. The presented FISH-TSA method provides strong signals without any interspecies reactivity.

Changes of femoral bone tissue microstructure in transgenic rabbits.

Bone tissue microstructure of femur was investigated in transgenic New Zealand White rabbits with human factor VIII gene. Altogether 42 bones (24 from transgenic rabbits and 18 from non-transgenic ones) were analysed. Specimens were prepared using standard histological equipment, producing thin sections of approximately 80-100 microm. For histomorphometrical analysis areas, perimeters, minimum and maximum diameters of osteons' vascular canals and of osteons were measured. We found out that the basic structural pattern of femoral bone tissue was primary vascular longitudinal in both groups of rabbits. However, a new type of the bone tissue--fibrolamellar--was identified only in the transgenic rabbits. The measured variables of the osteons' vascular canals were higher in transgenic individuals in comparison with the nontransgenic ones (except for maximal diameter) and the differences were statistically significant (P < 0.05; P < 0.01). We suppose that the observed differences could be associated with transgenesis. In an effort to explain these differences we compared the cytogenetic profile of bone marrow cells between transgenic and non-transgenic rabbits. A significantly higher rate of aneuploidy was observed in c-metaphase spreads of transgenic individuals as compared to non-transgenic ones (P < 0.001). Despite the fact that no hFVIII mRNA expression was found in the femur of transgenic rabbits, we discussed an association of transgene integration into the genome and microstructural changes in the bone. In any case, the results indicate that transgenesis can also produce changes in other tissues than in the target ones.

Evaluation of haematological, biochemical and histopathological parameters of transgenic rabbits.

The aim of our study was to compare the hFVIII mRNA expression in different organs, pathological changes and selected haematological and biochemical blood parameters between transgenic and non-transgenic rabbits from F3 generation. Selected physiological parameters of 3- to 4-month-old transgenic rabbits from F3 generation carrying human factor VIII gene (hFVIII) were analysed and compared with those of non-transgenic ones. Before slaughtering, the blood for haematological and biochemical analysis was taken from the central ear artery. Pathological and histological examination of vital organs and RT-PCR analysis of several tissue organs of transgenic and non-transgenic animals were performed after slaughtering. Except for the mammary gland tissue, slight hfVIII mRNA expression in the spleen, lung and brain and none expression in the liver, kidney, skeletal muscle and heart of rabbits were recorded. pathological examination of vital organs showed some pathological changes in both transgenic and non-transgenic rabbits which were confirmed by histological qualitative evaluations. Statistically significant lower values of blood haemoglobin in blood of transgenic (11.86+/-0.86) animals compared with non-transgenic (12.41+/-1.02, P<0.05) ones and lower parameters of HCT (39.22+/-2.44 versus 40.89+/-2.26, P<0.01) in blood of transgenic rabbits were observed. Parameters of WBC, RBC and PLT showed no significant differences between the analysed groups. All biochemical serum parameters of transgenic rabbits were higher in comparison with non-transgenic ones. Significant differences were found in the concentration of the urea, AST and GMT between transgenic and non-transgenic animals (P<0.001) and in the total protein content, the difference was significant at P<0.05. In conclusion, our results showed that no considerable impact on the general health was found in transgenic rabbits.

Comparative study of compact bone tissue microstructure between non-transgenic and transgenic rabbits with WAP-hFVIII gene construct.

The aim of this study was to investigate differences in compact bone tissue microstructure between New Zealand White transgenic rabbits with human blood clotting factor VIII gene and the non-transgenic ones. The transgene was under the transcriptional control of the whey acidic protein (WAP) promoter. Altogether, 42 femora were analysed. Specimens were prepared using standard histological equipment, producing thin sections approximately 80-100 microm. Areas, perimeters, minimum and maximum diameters of vascular canals of primary osteons, Haversian canals and secondary osteons were measured. Our results indicate the basic structural pattern of the bone tissue was primary vascular longitudinal in both groups of rabbits. However, a fibrolamellar bone tissue was identified only in the transgenic rabbits. The measured variables of the vascular canals of the primary osteons, the Haversian canals and the secondary osteons were higher in transgenic rabbits in comparison with non-transgenic ones in most cases and the differences were often statistically significant (P < 0.01; P < 0.001). More significant differences were noticed in males (transgenic versus non-transgenic). However, it is not easy to find the real cause of the changes. Despite the fact that no hFVIII mRNA expression was found in the analysed bone of transgenic rabbits, we presume that the observed differences could be associated with transgenesis. In this article, we discuss possible associations between transgene integration as well as aneuploidy and/or observed microstructural changes in compact bone tissue.