Mechanisms of Prostate Tumorigenesis Using Genetically Engineered Mouse Models

使用基因工程小鼠模型研究前列腺肿瘤发生机制

• 批准号: 7965790
• 负责人: Lino Tessarollo
• 金额: $ 64.45万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7965790
• 关键词: 3-Dimensional; Address; Age; Age-Months; Aging-Related Process; Alleles; Androgens; Animal Model; Appearance; Bacterial Artificial Chromosomes; Basal Cell; Binding; Cell Line; Cells; Coculture Techniques; Code; Complex; Data Collection; Development; Diagnostic tests; Epithelial; Epithelial-Stromal Communication; Epithelium; Evaluation; Evolution; Formalin; Genes; Genetically Engineered Mouse; Goals; Growth; Hand; Helicobacter; Histology; Hormones; Human; Hyperplasia; In Vitro; K-18 conjugate; KRT19 gene; Keratin; Knock-out; Lesion; Malignant - descriptor; Malignant Neoplasms; Malignant neoplasm of prostate; Modeling; Molecular; Molecular Abnormality; Molecular Analysis; Mouse, Founder, Transgenic; Mus; Mutation; Oils; Pathway interactions; Patients; Phenotype; Positioning Attribute; Predisposition; Process; Prostate; Prostatic Neoplasms; Proteins; Quarantine; RBL2 gene; Refractory Disease; Regulation; Regulatory Element; Relative (related person); Reporter; Role; SV40 T Antigens; Shipping; Ships; Signal Transduction; Site; Staging; Stromal Cells; System; TP53 gene; Tamoxifen; Technology; Tissue Harvesting; Tissues; Transgenes; Transgenic Mice; Tumor Suppression; Tumor Suppressor Proteins; Viral; Withdrawal; aged; androgen independent prostate cancer; cancer therapy; cell type; effective therapy; in vivo; interest; mouse model; neoplastic cell; p107 protein; pre-clinical; recombinase; response; transgene expression; tumor; tumor initiation; tumor progression; tumorigenesis; tumorigenic

Cell type susceptibility and androgen independence: The pRb tumor suppressor pathway is frequently altered in human prostate cancer. Our previous studies in multiple cell types have demonstrated the importance of pRb in suppressing tumor initiation. We have developed a strategy to dominantly interfere with pRb and potentially compensatory related proteins p107 and p130 by cell specific expression of the SV40 T antigen domain (T121) that binds to and inactivates all three proteins. We utilized keratin regulation to generate transgenic mice with androgen-independent epithelial subtype- targeted expression of T121 in prostate epithelium. In prostate, basal cells express K5 and K14, while luminal cells express K8 and K18. More recently, intermediate cells have been described with co expression of K5/18 and other keratins such as K15, 17 and 19. To explore the susceptible cell target(s), we used compartment-specific keratin gene transcriptional signals to drive expression of a conditional T121 allele composed of a reporter eGFP gene and stop sequences flanked by loxP sites upstream of T121 coding sequences. Specifically, K5, K18, and K19 genes were chosen to target basal cells, luminal cells, and intermediate cells in PE, respectively. To obtain optimal transgene expression, we have chosen to utilize a bacterial artificial chromosome (BAC) harboring the keratin gene to generate transgenic mice. The use of BAC-trangene minimizes position effects associated with standard transgenes and increases the likelihood that all necessary regulatory elements will be present. The eGFP stop cassette and T121 gene will be inserted into the BACs using recombineering technology pioneered by Neal Copeland and colleagues, such that regulators of the keratin gene will also drive the expression of a conditional T121 allele. The subsequent introduction of Cre recombinase (via somatic viral delivery or a tissue-specific transgene) will remove the stop sequences and induce T121 expression. All three strains of KeGT121 transgenic founder mice have been successfully generated (K5eGT121, K18eGT121, and K19eGT121). F1s have been used for mouse line characterization. Mice were also crossed to PbCre4 to activate T121 expression. To determine if tumor phenotype can be accelerated on Pten deficient background, we have crossed the mice on conditional Pten background. Year 2008, Van Dyke lab was in a transition from UNC-Chapel Hill to NCI-Frederick. Mouse colony has been shipped from UNC to NCI-Frederick. We are in the processing of rederiving all the single strains to helicobacter free facility. These strains are: K5eGT121 (4 lines), K18eGT121 (2 lines), K19eGT121 (2 lines), PbCre4 (1 line), conditional Pten (1 line). All the experimental mice are still in quarantine currently. Mice with tumors have been dissected and tissues were collected in either 10% formalin, OCT, or -80C. We are in the process of histology data collection. Stomal contribution to prostate tumor progression in APT121 mouse model: APT121;p53cf/+;FSPCreER, APT121;p53cf/f;FSPCreER, and relative control (e.g. p53cf/f;FSPCreER, p53cf/+;FSPCreE, and APT121) mice have been successfully generated. To inactivate p53, we i.p. injected the mice with tamoxifen (1mg/mouse/day) or oil as control at 2 months of age for 5 consecutive days. Mice are aged to various stages. Tissues are harvested for histological evaluation and molecular analysis. In year 2008, Van Dyke lab was in a transition from UNC-Chapel Hill to NCI-Frederick. Mouse colony has been shipped from UNC to NCI-Frederick. All the experimental mice are still in quarantine currently. Mice with tumors have been dissected and tissues were collected in either 10% formalin, OCT, or -80C. We are in the process of aging the mice, and collecting tissue samples.Cell type susceptibility and androgen independence: The pRb tumor suppressor pathway is frequently altered in human prostate cancer. Our previous studies in multiple cell types have demonstrated the importance of pRb in suppressing tumor initiation. We have developed a strategy to dominantly interfere with pRb and potentially compensatory related proteins p107 and p130 by cell specific expression of the SV40 T antigen domain (T121) that binds to and inactivates all three proteins. We utilized keratin regulation to generate transgenic mice with androgen-independent epithelial subtype- targeted expression of T121 in prostate epithelium. In prostate, basal cells express K5 and K14, while luminal cells express K8 and K18. More recently, intermediate cells have been described with co expression of K5/18 and other keratins such as K15, 17 and 19. To explore the susceptible cell target(s), we used compartment-specific keratin gene transcriptional signals to drive expression of a conditional T121 allele composed of a reporter eGFP gene and stop sequences flanked by loxP sites upstream of T121 coding sequences. Specifically, K5, K18, and K19 genes were chosen to target basal cells, luminal cells, and intermediate cells in PE, respectively. To obtain optimal transgene expression, we have chosen to utilize a bacterial artificial chromosome (BAC) harboring the keratin gene to generate transgenic mice. The use of BAC-trangene minimizes position effects associated with standard transgenes and increases the likelihood that all necessary regulatory elements will be present. The eGFP stop cassette and T121 gene will be inserted into the BACs using recombineering technology pioneered by Neal Copeland and colleagues, such that regulators of the keratin gene will also drive the expression of a conditional T121 allele. The subsequent introduction of Cre recombinase (via somatic viral delivery or a tissue-specific transgene) will remove the stop sequences and induce T121 expression. All three strains of KeGT121 transgenic founder mice have been successfully generated (K5eGT121, K18eGT121, and K19eGT121). F1s have been used for mouse line characterization. Mice were also crossed to PbCre4 to activate T121 expression. To determine if tumor phenotype can be accelerated on Pten deficient background, we have crossed the mice on conditional Pten background. Year 2008, Van Dyke lab was in a transition from UNC-Chapel Hill to NCI-Frederick. Mouse colony has been shipped from UNC to NCI-Frederick. We are in the processing of rederiving all the single strains to helicobacter free facility. These strains are: K5eGT121 (4 lines), K18eGT121 (2 lines), K19eGT121 (2 lines), PbCre4 (1 line), conditional Pten (1 line). All the experimental mice are still in quarantine currently. Mice with tumors have been dissected and tissues were collected in either 10% formalin, OCT, or -80C. We are in the process of histology data collection. Stomal contribution to prostate tumor progression in APT121 mouse model: APT121;p53cf/+;FSPCreER, APT121;p53cf/f;FSPCreER, and relative control (e.g. p53cf/f;FSPCreER, p53cf/+;FSPCreE, and APT121) mice have been successfully generated. To inactivate p53, we i.p. injected the mice with tamoxifen (1mg/mouse/day) or oil as control at 2 months of age for 5 consecutive days. Mice are aged to various stages. Tissues are harvested for histological evaluation and molecular analysis. In year 2008, Van Dyke lab was in a transition from UNC-Chapel Hill to NCI-Frederick. Mouse colony has been shipped from UNC to NCI-Frederick. All the experimental mice are still in quarantine currently. Mice with tumors have been dissected and tissues we [summary truncated at 7800 characters]

细胞类型易感性和雄激素独立性：pRb 肿瘤抑制途径在人类前列腺癌中经常发生改变。我们之前对多种细胞类型的研究已经证明了 pRb 在抑制肿瘤发生方面的重要性。我们开发了一种策略，通过 SV40 T 抗原结构域 (T121) 的细胞特异性表达来显性干扰 pRb 和潜在补偿性相关蛋白 p107 和 p130，该结构域与所有三种蛋白结合并使其失活。我们利用角蛋白调节来产生转基因小鼠，其在前列腺上皮中具有不依赖于雄激素的上皮亚型靶向表达 T121。在前列腺中，基底细胞表达 K5 和 K14，而管腔细胞表达 K8 和 K18。最近，中间细胞被描述为 K5/18 和其他角蛋白（如 K15、17 和 19）的共表达。为了探索易感细胞靶标，我们使用区室特异性角蛋白基因转录信号来驱动条件 T121 等位基因的表达，该等位基因由报告 eGFP 基因和终止序列组成，两侧是 T121 编码序列上游的 loxP 位点。具体来说，选择K5、K18和K19基因分别靶向PE中的基底细胞、管腔细胞和中间细胞。为了获得最佳的转基因表达，我们选择利用含有角蛋白基因的细菌人工染色体（BAC）来产生转基因小鼠。 BAC-转基因的使用最大限度地减少了与标准转基因相关的位置效应，并增加了所有必要的调控元件存在的可能性。 eGFP 终止盒和 T121 基因将使用 Neal Copeland 及其同事首创的重组工程技术插入 BAC 中，这样角蛋白基因的调节因子也将驱动条件 T121 等位基因的表达。 随后引入 Cre 重组酶（通过体细胞病毒递送或组织特异性转基因）将去除终止序列并诱导 T121 表达。所有三种 KeGT121 转基因创始小鼠品系均已成功生成（K5eGT121、K18eGT121 和 K19eGT121）。 F1 已用于小鼠品系表征。小鼠还与 PbCre4 杂交以激活 T121 表达。为了确定肿瘤表型是否可以在 Pten 缺陷背景下加速，我们在条件 Pten 背景下对小鼠进行了杂交。 2008 年，Van Dyke 实验室从 UNC-Chapel Hill 过渡到 NCI-Frederick。 小鼠群体已从 UNC 运送到 NCI-Frederick。我们正在将所有单一菌株重新衍生到无螺杆菌设施中。这些菌株是：K5eGT121（4 系）、K18eGT121（2 系）、K19eGT121（2 系）、PbCre4（1 系）、条件 Pten（1 系）。目前所有实验小鼠仍处于隔离状态。解剖带有肿瘤的小鼠并在 10% 福尔马林、OCT 或 -80C 中收集组织。我们正在收集组织学数据。 APT121 小鼠模型中造口对前列腺肿瘤进展的贡献：APT121;p53cf/+;FSPCreER、APT121;p53cf/f;FSPCreER 和相对对照（例如 p53cf/f;FSPCreER、p53cf/+;FSPCreE 和 APT121）小鼠已成功生成。为了灭活 p53，我们进行 i.p.小鼠2月龄时注射他莫昔芬（1mg/小鼠/天）或油作为对照，连续5天。小鼠衰老到不同的阶段。收获组织用于组织学评估和分子分析。 2008 年，Van Dyke 实验室从 UNC-Chapel Hill 过渡到 NCI-Frederick。小鼠群体已从 UNC 运送到 NCI-Frederick。目前所有实验小鼠仍处于隔离状态。解剖带有肿瘤的小鼠并在 10% 福尔马林、OCT 或 -80C 中收集组织。我们正在使小鼠老化并收集组织样本。细胞类型敏感性和雄激素独立性：pRb 肿瘤抑制途径在人类前列腺癌中经常发生改变。我们之前对多种细胞类型的研究已经证明了 pRb 在抑制肿瘤发生方面的重要性。我们开发了一种策略，通过 SV40 T 抗原结构域 (T121) 的细胞特异性表达来显性干扰 pRb 和潜在补偿性相关蛋白 p107 和 p130，该结构域与所有三种蛋白结合并使其失活。我们利用角蛋白调节来产生转基因小鼠，其在前列腺上皮中具有不依赖于雄激素的上皮亚型靶向表达 T121。在前列腺中，基底细胞表达 K5 和 K14，而管腔细胞表达 K8 和 K18。最近，中间细胞被描述为 K5/18 和其他角蛋白（如 K15、17 和 19）的共表达。为了探索易感细胞靶标，我们使用区室特异性角蛋白基因转录信号来驱动条件 T121 等位基因的表达，该等位基因由报告 eGFP 基因和终止序列组成，两侧是 T121 编码序列上游的 loxP 位点。具体来说，选择K5、K18和K19基因分别靶向PE中的基底细胞、管腔细胞和中间细胞。为了获得最佳的转基因表达，我们选择利用含有角蛋白基因的细菌人工染色体（BAC）来产生转基因小鼠。 BAC-转基因的使用最大限度地减少了与标准转基因相关的位置效应，并增加了所有必要的调控元件存在的可能性。 eGFP 终止盒和 T121 基因将使用 Neal Copeland 及其同事首创的重组工程技术插入 BAC 中，这样角蛋白基因的调节因子也将驱动条件 T121 等位基因的表达。 随后引入 Cre 重组酶（通过体细胞病毒递送或组织特异性转基因）将去除终止序列并诱导 T121 表达。所有三种 KeGT121 转基因创始小鼠品系均已成功生成（K5eGT121、K18eGT121 和 K19eGT121）。 F1 已用于小鼠品系表征。小鼠还与 PbCre4 杂交以激活 T121 表达。为了确定肿瘤表型是否可以在 Pten 缺陷背景下加速，我们在条件 Pten 背景下对小鼠进行了杂交。 2008 年，Van Dyke 实验室从 UNC-Chapel Hill 过渡到 NCI-Frederick。 小鼠群体已从 UNC 运送到 NCI-Frederick。我们正在将所有单一菌株重新衍生到无螺杆菌设施中。这些菌株是：K5eGT121（4 系）、K18eGT121（2 系）、K19eGT121（2 系）、PbCre4（1 系）、条件 Pten（1 系）。目前所有实验小鼠仍处于隔离状态。解剖带有肿瘤的小鼠并在 10% 福尔马林、OCT 或 -80C 中收集组织。我们正在收集组织学数据。 APT121 小鼠模型中造口对前列腺肿瘤进展的贡献：APT121;p53cf/+;FSPCreER、APT121;p53cf/f;FSPCreER 和相对对照（例如 p53cf/f;FSPCreER、p53cf/+;FSPCreE 和 APT121）小鼠已成功生成。为了灭活 p53，我们进行 i.p.小鼠2月龄时注射他莫昔芬（1mg/小鼠/天）或油作为对照，连续5天。小鼠衰老到不同的阶段。收获组织用于组织学评估和分子分析。 2008 年，Van Dyke 实验室从 UNC-Chapel Hill 过渡到 NCI-Frederick。小鼠群体已从 UNC 运送到 NCI-Frederick。目前所有实验小鼠仍处于隔离状态。患有肿瘤的小鼠已被解剖，我们的组织[摘要被截断为 7800 个字符]