67 PRODUCTION OF CLONED TRANSGENIC RABBITS FROM MESENCHYMAL STEM CELLS

Abstract

Mesenchymal stem cells (MSC) are adult stem cells with fibroblast-like morphology, which can be easily isolated from bone marrow and expanded in culture. Mesenchymal stem cells are able to grow from a single cell into a cell clone, which makes them potentially useful for gene targeting. In our recent study we investigated the dynamics of epigenetic reprogramming following nuclear transfer (NT) with MSC and found that these cells can support development of cloned embryos as good as genetically identical fibroblasts (Brero et al. 2009 Cloning Stem Cells 11, 319-329). In the present study we tested whether live cloned rabbits can be produced from MSC. Nuclear donor cells were isolated from a 6-week-old transgenic Ali/Bas rabbit, expanded in culture, and assessed for their differentiation potential. Mesenchymal stem cells were transfected with a green fluorescent protein (GFP) reporter gene construct and stable cell clones were selected (GFP-MSC). The MSC and GFP-MSC were used for NT at passage 3 to 7 after serum starvation for 2 to 4 days. Nuclear transfer was performed essentially as described previously (Yang et al. 2007 Reproduction 133, 219-320). To assess the development to blastocyst, reconstructed embryos were cultured in B2 medium for 5 to 6 days, whereas for in vivo development embryos were cultured only overnight and then transferred into recipients at the 4- to 8-cell stage. In the MSC group, 844 oocytes were used, 793 (94%) of them fused, 698/786 (89%) cleaved, and 48/128 (38%) developed to blastocyst. After transfer of 483 cloned embryos into 13 recipients, 2 from 8 pregnant recipients gave birth to 10 (2.4%) rabbits, from which 2 and 1 survived for more than 7 days and 3 months, respectively. In the GFP-MSC group, 444 oocytes were used, 412 (93%) of them fused, 377/409 (92%) cleaved, and 97/178 (55%) developed to blastocyst. Transfer of 216 cloned embryos into 8 recipients resulted in 4 pregnancies. One recipient gave birth to 6 (3.7%) live and 2 stillborn rabbits, from which 2 and 1 rabbits survived for more than 3 days and 2 weeks, respectively. All cloned rabbits carried a GFP gene, and green fluorescence could be detected in the follicles of the skin under a fluorescence microscope (Zeiss Axiovert200, Carl Zeiss, Germany). Our study demonstrates that live cloned rabbits can be produced from genetically modified MSC, thus paving the way to generate gene targeted animals.

This work is supported by Roche Diagnostic GmbH.