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

journal contribution
posted on 06.12.2017, 00:00 by Y Luo, J Li, C Sorensen, Y Liu, L Lin, Y Du, S Li, H Yang, Gabor Vajta, H Callesen
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?/D11), and one as wild type. The BRCA1 expression was decreased at the mRNA level in BRCA1?/D11 fibroblasts. However, all BRCA1?/D11 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.

Funding

Category 1 - Australian Competitive Grants (this includes ARC, NHMRC)

History

Volume

20

Issue

5

Start Page

975

End Page

988

Number of Pages

14

eISSN

1573-9368

ISSN

0962-8819

Location

Netherlands

Publisher

Springer Netherlands

Language

en-aus

Peer Reviewed

Yes

Open Access

No

External Author Affiliations

Aarhus universitet; BGI-Shenzhen; TBA Research Institute;

Era Eligible

Yes

Journal

Transgenic research.

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