在过去的二十年中,已经使用了强大的组成型和诱导型乳腺癌转基因/双基因/三基因小鼠模型来阐明给定的转基因癌基因与其他细胞基因相互作用并开始运动的分子机制。乳腺癌的开始和进展。然而,这些转基因模型,仅作为体内模型,它们提供的操纵实验变量的机会是昂贵且限制性的,这些变量将能够更好地理解与初始转化和正在转化的靶细胞相关的分子事件。
为了克服这些限制,我们从这些转基因小鼠的尾静脉成纤维细胞中获得了含有致癌基因的诱导多能干细胞(iPSC)克隆,并在非转基因背景小鼠中进行了体外和体内操作。我们创建了具有相对较低M.O.I的iPSC克隆,产生逆转录病毒整合,平均每个逆转录病毒质粒构建体仅使用1至2个位点。
来自每个组的大多数iPSC克隆显示出基本正常的鼠核型,外源重编程基因的强表达和特征性内源性鼠表面干细胞标志物包括SSEA-1(CD15)的显著表达,PECAM-1(CD31),Ep-Cam(CD326),和Nectin(CD112),但不表达他们的转基因。大多数(75%)iPSC克隆显示正常鼠核型,但25%显示基因组不稳定核型。然而,即使这些后来的克隆也表现出稳定和特征性的iPSC特性。当原位注射时,选择iPSC克隆,组成型或诱导型,不再表达其外源性多能性重编程因子,而是表达其致癌转基因(PyVT或ErbB2),并参与乳腺个体发育和随后的肿瘤发生。当非原位注射或沿几种不同的非乳腺分化谱系在体外分化时,iPSC克隆未能这样做。尽管许多克隆产生了预期的畸胎瘤,选择iPSC克隆在适当的组成型或诱导型条件下表现出乳腺个体发育和肿瘤发生通过相同的阶段,表现出他们的转基因鼠亲本和,因此,代表转基因替代品。与转基因小鼠相比,iPSC克隆提供了许多优势,包括成本,在体外操纵和标记的能力,并创建乳腺个体发育和肿瘤发生的体外模型,该模型可用于获得对易于转化的靶细胞分化状态的更多见解。此外,这些含有癌基因的iPSC克隆可用于乳腺癌的化学预防研究.
Powerful constitutive and inducible transgenic / bitransgenic / tritransgenic murine models of breast cancer have been used over the past two decades to shed light on the molecular mechanisms by which the given transgenic oncogenes have interacted with other cellular genes and set in motion breast cancer initiation and progression. However, these transgenic models, as in vivo models only, are expensive and restrictive in the opportunities they provide to manipulate the experimental variables that would enable a better understanding of the molecular events related to initial transformation and the target cell being transformed.
To overcome some of these limitations, we derived oncogene-containing induced pluripotent stem cell (iPSC) clones from tail vein fibroblasts of these transgenic mice and manipulated them both in vitro and in vivo in non-transgenic background mice. We created the iPSC clones with a relatively low M.O.I, producing retroviral integrations which averaged only 1 to 2 sites per retroviral plasmid construct used.
Most iPSC clones derived from each group displayed an essentially normal murine karyotype, strong expression of the exogenous reprogrammable genes and significant expression of characteristic endogenous murine surface stem cell markers including SSEA-1 (CD15), PECAM-1 (CD31), Ep-Cam (CD326), and Nectin (CD112), but no expression of their transgene. A majority (75%) of iPSC clones displayed a normal murine karyotype but 25% exhibited a genomically unstable karyotype. However, even these later clones exhibited stable and characteristic iPSC properties. When injected orthotopically, select iPSC clones, either constitutive or inducible, no longer expressed their exogenous pluripotency reprogramming factors but expressed their oncogenic transgene (PyVT or ErbB2) and participated in both breast ontogenesis and subsequent oncogenesis. When injected non-orthotopically or when differentiated in vitro along several different non-mammary lineages of differentiation, the iPSC clones failed to do so. Although many clones developed anticipated teratomas, select iPSC clones under the appropriate constitutive or inducible conditions exhibited both breast ontogenesis and oncogenesis through the same stages as exhibited by their transgenic murine parents and, as such, represent transgenic surrogates.
The iPSC clones offer a number of advantages over transgenic mice including cost, the ability to manipulate and tag in vitro, and create an in vitro model of breast ontogeny and oncogenesis that can be used to gain additional insights into the differentiated status of the target cell which is susceptible to transformation. In addition, the use of these oncogene-containing iPSC clones can be used in chemopreventive studies of breast cancer.