Shingu Takashi, Jaskelioff Mariela, Yuan Liang, Ding Zhihu, Protopopov Alexei, Kost-Alimova Maria, Hu Jian
Department of Cancer Biology, UT MD Anderson Cancer Center.
Novartis Institutes for Biomedical Research.
J Vis Exp. 2015 Apr 13(98):52599. doi: 10.3791/52599.
Telomere dysfunction-induced loss of genome integrity and its associated DNA damage signaling and checkpoint responses are well-established drivers that cause tissue degeneration during ageing. Cancer, with incidence rates greatly increasing with age, is characterized by short telomere lengths and high telomerase activity. To study the roles of telomere dysfunction and telomerase reactivation in ageing and cancer, the protocol shows how to generate two murine inducible telomerase knock-in alleles 4-Hydroxytamoxifen (4-OHT)-inducible TERT-Estrogen Receptor (mTERT-ER) and Lox-Stopper-LoxTERT (LSL-mTERT). The protocol describes the procedures to induce telomere dysfunction and reactivate telomerase activity in mTERT-ER and LSL-mTERT mice in vivo. The representative data show that reactivation of telomerase activity can ameliorate the tissue degenerative phenotypes induced by telomere dysfunction. In order to determine the impact of telomerase reactivation on tumorigenesis, we generated prostate tumor model G4 PB-Cre4 Pten(L/L) p53(L/L) LSL-mTERT(L/L) and thymic T-cell lymphoma model G4 Atm(-/-) mTERT(ER/ER). The representative data show that telomerase reactivation in the backdrop of genomic instability induced by telomere dysfunction can greatly enhance tumorigenesis. The protocol also describes the procedures used to isolate neural stem cells (NSCs) from mTERT-ER and LSL-mTERT mice and reactivate telomerase activity in NSCs in vitro. The representative data show that reactivation of telomerase can enhance the self-renewal capability and neurogenesis in vitro. Finally, the protocol describes the procedures for performing telomere FISH (Fluorescence In Situ Hybridization) on both mouse FFPE (Formalin Fixed and Paraffin Embedded) brain tissues and metaphase chromosomes of cultured cells.
端粒功能障碍导致的基因组完整性丧失及其相关的DNA损伤信号传导和检查点反应是公认的导致衰老过程中组织退化的驱动因素。癌症的发病率随着年龄的增长而大幅上升，其特征是端粒长度短和端粒酶活性高。为了研究端粒功能障碍和端粒酶重新激活在衰老和癌症中的作用，本方案展示了如何生成两种小鼠诱导型端粒酶敲入等位基因——4-羟基他莫昔芬（4-OHT）诱导型TERT-雌激素受体（mTERT-ER）和Lox-Stopper-LoxTERT（LSL-mTERT）。该方案描述了在体内诱导mTERT-ER和LSL-mTERT小鼠端粒功能障碍并重新激活端粒酶活性的程序。代表性数据表明，端粒酶活性的重新激活可以改善端粒功能障碍诱导的组织退化表型。为了确定端粒酶重新激活对肿瘤发生的影响，我们构建了前列腺肿瘤模型G4 PB-Cre4 Pten(L/L) p53(L/L) LSL-mTERT(L/L)和胸腺T细胞淋巴瘤模型G4 Atm(-/-) mTERT(ER/ER)。代表性数据表明，在端粒功能障碍诱导的基因组不稳定背景下，端粒酶重新激活可极大地增强肿瘤发生。该方案还描述了从mTERT-ER和LSL-mTERT小鼠中分离神经干细胞（NSC）并在体外重新激活NSC中端粒酶活性的程序。代表性数据表明，端粒酶重新激活可增强体外自我更新能力和神经发生。最后，该方案描述了对小鼠福尔马林固定石蜡包埋（FFPE）脑组织和培养细胞中期染色体进行端粒荧光原位杂交（FISH）的程序。
