title: High efficiency of BRCA1 knockout using rAAV-mediated gene targeting: developing a pig model for breast cancer.
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.
authors.affiliation: Department of Human Genetics, Aarhus University, Wilhelm Meyers Alle 4, 8000 Aarhus C, Denmark. alun@humgen.au.dk
authors.forename: Yonglun
authors.initials: Y
authors.lastname: Luo
authors.forename: Juan
authors.initials: J
authors.lastname: Li
authors.forename: Ying
authors.initials: Y
authors.lastname: Liu
authors.forename: Lin
authors.initials: L
authors.lastname: Lin
authors.forename: Yutao
authors.initials: Y
authors.lastname: Du
authors.forename: Shengting
authors.initials: S
authors.lastname: Li
authors.forename: Huanming
authors.initials: H
authors.lastname: Yang
authors.forename: Gábor
authors.initials: G
authors.lastname: Vajta
authors.forename: Henrik
authors.initials: H
authors.lastname: Callesen
authors.forename: Lars
authors.initials: L
authors.lastname: Bolund
authors.forename: Charlotte Brandt
authors.initials: CB
authors.lastname: Sørensen
mesh_terms: D000818:Animals; D019313:BRCA1 Protein / Q000235:genetics*; D001943:Breast Neoplasms / Q000235:genetics*; D000229:Dependovirus / Q000235:genetics; D004195:Disease Models, Animal; D004624:Embryo Transfer; D005260:Female; D055786:Gene Knockout Techniques; D018390:Gene Targeting; D006579:Heterozygote; D010802:Phylogeny; D013552:Swine / Q000235:genetics* / Q000378:metabolism; D013556:Swine, Miniature / Q000235:genetics* / Q000378:metabolism
chemical_list: D019313:BRCA1 Protein