CSIRO Publishing blank image blank image blank image blank imageBooksblank image blank image blank image blank imageJournalsblank image blank image blank image blank imageAbout Usblank image blank image blank image blank imageShopping Cartblank image blank image blank image You are here: Journals > Reproduction, Fertility and Development   
Reproduction, Fertility and Development
Journal Banner
  Vertebrate reproductive science and technology
blank image Search
blank image blank image
blank image
  Advanced Search

Journal Home
About the Journal
Editorial Structure
Online Early
Current Issue
Just Accepted
All Issues
Special Issues
Research Fronts
Virtual Issues
Sample Issue
For Authors
General Information
Submit Article
Author Instructions
Open Access
Awards and Prizes
For Referees
Referee Guidelines
Review an Article
Annual Referee Index
For Subscribers
Subscription Prices
Customer Service
Print Publication Dates
Library Recommendation

blue arrow e-Alerts
blank image
Subscribe to our email Early Alert or RSS feeds for the latest journal papers.

red arrow Connect with us
blank image
facebook twitter logo LinkedIn

red arrow Connect with SRB
blank image
facebook TwitterIcon

Affiliated Societies

RFD is the official journal of the International Embryo Transfer Society and the Society for Reproductive Biology.


Article << Previous     |     Next >>   Contents Vol 23(1)


K. Wallner A, A. Wuensch A, K. Burkhardt A, M. Kurome A, B. Kessler A, P. Fezert A, A. Richter A, H. Nagashima B, N. Klymiuk A and E. Wolf A

A Chair for Molecular Animal Breeding and Biotechnology, LMU Munich, Munich, Bavaria, Germany;
B Meiji University, Kawasaki, Japan

Reproduction, Fertility and Development 23(1) 264-265 http://dx.doi.org/10.1071/RDv23n1Ab338
Published: 7 December 2010

 Export Citation

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.

Supported by the Mukoviszidose e.V. and the Bayerische Forschungsstiftung.

Legal & Privacy | Contact Us | Help


© CSIRO 1996-2016