Are you K Wallner?

Claim your profile

Publications (2)5.17 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Cystic fibrosis (CF), the most common inherited disease in the Caucasian population, is caused by mutations of the CFTR gene, coding for an anion channel responsible for chloride movement, determining transepithelial salt transport, fluid flow and ion concentrations. Cystic fibrosis is determined by chronic lung disease, marked by airway surface dehydration, reduced mucus clearance and increased mucus obstruction, impaired bacterial killing and inflammation and continuous destruction of the lung tissue. The lack of suitable CF mouse models has so far impeded the understanding of disease mechanisms and the development of novel therapies. Recently, a pig model reflecting the main hallmarks of the human disease in the respiratory, gastrointestinal and genital tracts has been reported (Rogers et al. 2008 Science 321, 1837-1841). In the present study, we generated a new CF pig model by disrupting the CFTR gene using an approach that differs in 3 major aspects from the previously published model: (1) gene targeting was achieved using a modified bacterial artificial chromosome (BAC) vector; (2) transcription of the CFTR gene was completely prevented; and (3) homozygous CFTR mutant pigs were derived by nuclear transfer from cells in which both alleles were inactivated by sequential BAC targeting. We characterised 2 cloned litters with a total of 13 piglets (including 4 stillborns). All piglets showed a characteristic CF multi-organ disease with severe meconium ileus, microcolon, microgallbladder, hypoplasia of the exocrine pancreas, congenital aplasia of the vas deferens and malformations of the trachea. In contrast to the recently published CF pig, where the majority of the intestinal obstructions occurred proximal to the ileocecal junction, we found obstructions exclusively distal to this site. We thus present a novel pig model for CF that reflects the main hallmarks of the human disease. It confirms the suitability of the CFTR mutant pig for cystic fibrosis research and increases the availability of CF models for molecular and translational CF research.
    Reproduction Fertility and Development 12/2011; 24(1):232. · 2.58 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Site-directed mutagenesis provided a powerful tool for studying gene functions in mice, but the lack of embryonic stem cells in other species limited the application of this technology to other species. Various attempts using negative selection, viral vectors, or other auxiliary means promoted specific projects but did not provide methods for routine experiments. Here, we describe a novel approach that enabled the site-directed modification of 3 different porcine genes relevant for biomedical research. Three main technologies were combined to achieve these goals: bacterial artificial chromosome (BAC) vectors, somatic cell transfection, and nuclear transfer (SCNT). BAC vectors contain large genomic regions in bacterial plasmids. They are superior to conventional targeting tools, as they provide extended regions of homology of several kilobases. Novel recombination tools using bacterial enzymes enable the modification of any DNA region of interest and thus allow the introduction of desired mutations into BACs. After verification of the wt-BAC sequence, it was altered by using modification vectors carrying the desired mutation. The modified BAC vectors are linearized and transfected after verification into primary kidney cell lines, and cells are selected for integration of the vectors. Kidney cells provide both good proliferation and high targeting rates, and thus improve the efficiency compared to fetal fibroblasts. Singularized clones are screened for the replacement of wild-type targeting loci by quantitative PCR. Targeted clones are used for SCNT and transfer of the resulting embryos into synchronized gilts. We have evaluated this technology by the modification of the porcine CFTR, GGTA1, and DMD genes. All 3 genes are relevant for biomedical research, as mutations in CFTR are causative for cystic fibrosis, the knockout of GGTA1 is essential for overcoming hyperacute rejection in xenotransplantation, and various deletions in the DMD gene are responsible for Duchenne muscular dystrophy. Gaining 13 targeted clones out of 1152 for CFTR, 9 out of 306 for GGTA1 and 6 out of 203 for DMD, we obtained efficiencies higher than 1% for each of the target genes. The power of our approach is underlined by the fact that CFTR and DMD are loci that are thought to be difficult to manipulate. The viability of targeted kidney cells and their suitability for nuclear transfer is accentuated by the pregnancy rates (2 out of 3) and the delivery of 4 to 10 piglets or fetuses in the case of CFTR and GGTA1. The heterozygous fetuses or piglets are verified by qPCR. In the case of the X-chromosomal DMD gene, we have generated the first full knockout by transfecting male cells. Pregnancies of a successfully targeted clone are under way. Thus, we consider the combination of modified BAC vectors, transfection of kidney cells, and nuclear transfer to be a technology with the potential for routine production of site-directed mutations.
    Reproduction Fertility and Development 01/2011; 23(1):264-265. · 2.58 Impact Factor