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High efficiency of BRCA1 knockout using rAAV-mediated gene targeting: developing a pig model for breast cancer

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Abstract

Germline inactivating mutations of the breast cancer associated gene 1 (BRCA1) predispose to breast cancer and account for most cases of familiar breast and/or ovarian cancer. The pig is an excellent model for medical research as well as testing of new methods and drugs for disease prevention and treatment. We have generated cloned BRCA1 knockout (KO) Yucatan miniature piglets by targeting exon 11 using recombinant adeno-associated virus (rAAV)-mediated gene targeting and somatic cell nuclear transfer by Handmade Cloning (HMC). We found a very high targeting rate of rAAV-mediated BRCA1 KO. Approximately 35% of the selected cells were BRCA1 targeted. One BRCA1 KO cell clone (5D1), identified by PCR and Southern blot, was used as nuclear donor for HMC. Reconstructed embryos were transferred to three recipient sows which gave birth to 8 piglets in total. Genotyping identified seven piglets as BRCA1 heterozygotes (BRCA1+/∆11), and one as wild type. The BRCA1 expression was decreased at the mRNA level in BRCA1+/∆11 fibroblasts. However, all BRCA1+/∆11 piglets died within 18 days after birth. The causes of perinatal mortality remain unclear. Possible explanations may include a combination of the BRCA1 haploinsufficiency, problems of epigenetic reprogramming, presence of the marker gene, single cell clone effects, and/or the special genetic background of the minipigs.

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Acknowledgments

We would like to thank Bert Vogelstein and Kenneth W. Kinzler, John Hopkins University, Baltimore, US, for kindly providing the pNeDaKO plasmid vector and Dr. R. Jude Samulski and the UNC Vector Core Facility, Chapel Hill, North Carolina, US for the rAAV packaging. We would also like to thank Lisbeth Dahl Schrøder, Tina Hindkjær, Anette Thomsen, Bodil Schmidt and Christian Knudsen for skilled technical assistance. The project was supported by grants from the “Pig and Health Platform” of the Danish National Advanced Technology Foundation (Højteknologifonden), the Danish Agency for Science, Technology and Innovation (grant no. 274-05-0535), the DAnish Genetically Modified Animal Ressource (DAGMAR), and the “Sino-Danish Breast Cancer Research Centre” under the auspices of the Danish National Research Foundation (Grundforskningsfonden) and the National Natural Science Foundation of China.

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Authors and Affiliations

  1. Department of Human Genetics, Aarhus University, Wilhelm Meyers Alle 4, 8000, Aarhus C, Denmark

    Yonglun Luo, Lars Bolund & Charlotte Brandt Sørensen

  2. Section of Population Genetics and Embryology, Department of Genetics and Biotechnology, Research Centre Foulum, Aarhus University, Blichers Alle, 8830, Tjele, Denmark

    Juan Li, Ying Liu, Lin Lin, Gábor Vajta & Henrik Callesen

  3. BGI Shenzhen (HuaDa Shenzhen), 518083, Shenzhen, China

    Yonglun Luo, Lin Lin, Yutao Du, Huanming Yang & Lars Bolund

  4. Bioinformatics Research Centre, Aarhus University, C.F. Møllers Allé, 8000, Aarhus C, Denmark

    Shengting Li

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  1. Yonglun Luo
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  2. Juan Li
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  3. Ying Liu
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  11. Charlotte Brandt Sørensen
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Correspondence to Yonglun Luo.

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Luo, Y., Li, J., Liu, Y. et al. High efficiency of BRCA1 knockout using rAAV-mediated gene targeting: developing a pig model for breast cancer. Transgenic Res 20, 975–988 (2011). https://doi.org/10.1007/s11248-010-9472-8

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  • DOI: https://doi.org/10.1007/s11248-010-9472-8

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