Department of Laboratory Animal Medicine, Research Institute for Veterinary Science, BK21 PLUS Program for Creative Veterinary Science Research, College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea.
Division of Integrated Sciences for Life, Graduate School of Integrated Sciences for Life, Hiroshima University, Hiroshima, Japan.
Prostate. 2021 Feb;81(3):182-193. doi: 10.1002/pros.24095. Epub 2020 Dec 28.
Recent developments in gene editing, using transcriptional activator-like effector nucleases (TALENs), have greatly helped the generation of genetically engineered animal models. The NK3 homeobox 1 (NKX3.1) protein plays important roles in prostate development and protein production, and functions as a tumor suppressor. Recently, NKX3.1 was shown to be associated with breast cancer in humans.
Our aim was to create a new rat model to elucidate the functions of NKX3.1. To that end, we generated Nkx3.1 knockout rats using TALENs and analyzed their phenotype. TALEN-mediated Nkx3.1 knockout was confirmed by T7 endonuclease I (T7E1) assay and DNA sequencing. Prostate weight and fertility were evaluated in the knockout rats, besides determining the proportion of epithelial cells and messenger RNA (mRNA) expression of genes associated with carcinogenesis. Breast tumors were examined by histopathology.
Results suggested Nkx3.1 knockout rats have reduced fertility, decreased prostate weights, and increased epithelial cell layers. The mRNA expression of genes related to prostate carcinogenesis, namely Ar, Akt, and Pi3k, also increased. Moreover, the Nkx3.1 knockout rats often developed malignant breast tumors.
We, therefore, successfully created the first Nkx3.1 knockout rat model, using TALEN-mediated gene targeting, and used it to identify defects associated with Nkx3.1 deficiency, not previously observed in mice. Loss of Nkx3.1 in rats led to lower reproductive capacity, and decreased prostate weights, apart from the risk of developing breast cancer. We, thus, proposed Nkx3.1 knockout rats as reliable models for studying the role of NKX3.1 in decreased prostate weights, fertility, and breast cancer, as well as in prostate cancer.
转录激活因子样效应物核酸酶（TALENs）的基因编辑技术的最新进展极大地帮助了基因工程动物模型的产生。NK3 同源盒 1（NKX3.1）蛋白在前列腺发育和蛋白质产生中发挥重要作用，并作为肿瘤抑制因子发挥作用。最近，NKX3.1 被证明与人类乳腺癌有关。
我们的目的是创建一种新的大鼠模型来阐明 NKX3.1 的功能。为此，我们使用 TALENs 生成了 Nkx3.1 敲除大鼠，并分析了它们的表型。TALEN 介导的 Nkx3.1 敲除通过 T7 内切酶 I（T7E1）测定和 DNA 测序得到确认。除了确定与致癌相关的基因的上皮细胞比例和信使 RNA（mRNA）表达外，还评估了敲除大鼠的前列腺重量和生育能力。通过组织病理学检查检查乳腺癌肿瘤。
结果表明，Nkx3.1 敲除大鼠的生育能力降低，前列腺重量降低，上皮细胞层增加。与前列腺癌发生相关的基因 Ar、Akt 和 Pi3k 的 mRNA 表达也增加。此外，Nkx3.1 敲除大鼠经常发生恶性乳腺肿瘤。
因此，我们使用 TALEN 介导的基因靶向成功创建了第一个 Nkx3.1 敲除大鼠模型，并利用该模型鉴定了与 Nkx3.1 缺失相关的缺陷，这些缺陷在小鼠中尚未观察到。大鼠 Nkx3.1 的缺失导致繁殖能力降低，前列腺重量降低，以及乳腺癌的风险增加。因此，我们提出 Nkx3.1 敲除大鼠是研究 NKX3.1 在降低前列腺重量、生育能力和乳腺癌以及前列腺癌中的作用的可靠模型。
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