High efficiency of BRCA1 knockout using rAAV-mediated gene targeting: developing a pig model for breast cancer
High efficiency of BRCA1 knockout using rAAV-mediated gene targeting: developing a pig model for breast cancer
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DOI：
10.1007/s11248-010-9472-8
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发表时间：
2011-10-01
影响因子：
3
通讯作者：
Sorensen, Charlotte Brandt
中科院分区：
文献类型：
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作者：
Luo, Yonglun;Li, Juan;Sorensen, Charlotte Brandt
乳腺癌相关基因1（BRCA 1）的生殖系失活突变易患乳腺癌，并导致大多数常见的乳腺癌和/或卵巢癌。猪是医学研究以及测试疾病预防和治疗的新方法和药物的极好模型。我们已经产生了克隆BRCA 1敲除（KO）尤卡坦小型猪通过使用重组腺相关病毒（rAAV）介导的基因打靶和体细胞核移植手工克隆（HMC）的外显子11为靶点。我们发现rAAV介导的BRCA 1 KO的靶向率非常高。大约35%的选定细胞是BRCA 1靶向的。将经PCR和Southern blot鉴定的一个BRCA 1 KO细胞克隆（5D 1）用作HMC的核供体。将重建的胚胎转移到三头受体母猪中，总共产下8头仔猪。基因分型鉴定7头仔猪为BRCA 1杂合子（BRCA 1（+/Delta 11）），1头为野生型。BRCA 1（+/Delta 11）成纤维细胞BRCA 1表达在mRNA水平降低。然而，所有BRCA 1（+/Delta 11）仔猪均在出生后18天内死亡。围产期死亡的原因尚不清楚。可能的解释包括BRCA 1单倍不足、表观遗传重编程问题、标记基因的存在、单细胞克隆效应和/或小型猪的特殊遗传背景。
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(+/Delta 11)), and one as wild type. The BRCA1 expression was decreased at the mRNA level in BRCA1(+/Delta 11) fibroblasts. However, all BRCA1(+/Delta 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.