SYNTHESIS AND FUNCTION OF A TRANSFORMATION-DEPENDENT SECRETED LYSOSOMAL PROTEASE

转化依赖性分泌型溶酶体蛋白酶的合成和功能

• 批准号: 5200962
• 负责人: M M GOTTESMAN
• 金额: --
• 依托单位: DIVISION OF CANCER BIOLOGY AND DIAGNOSIS
• 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/5200962
• 关键词: embryonic stem cell; enzyme biosynthesis; genetically modified animals; laboratory mouse; molecular cloning; protein structure function; secretion; transfection /expression vector; zymogens

Procathepsin L is the precursor to the lysosomal cysteine protease cathepsin L. It is secreted in large amounts by malignantly transformed mouse cells, and by cells treated with tumor promoters or growth factors. The function of procathepsin L in malignancy is not known, but involvement in tumor invasion and metastasis and in suppression of the immune response to tumors has been suggested. In normal tissues, procathepsin L is secreted by the liver, osteoclasts, and Sertoli cells, indicating possible involvement in bone resorption and sperm maturation. To study the biological function of procathepsin L, we are attempting to inactivate this gene by insertional mutagenesis into embryonic stem (ES) cells with the goal of establishing transgenic mice lacking cathepsin L function. We have cloned cathepsin L DNA isogenic with ES cell DNA into two targeting vectors (pPNT(s) and pSSC9) which carry different promoters and different numbers of Herpes Simplex Virus (HSV) thymidine kinase (tk) genes. The pSSC9 vector was constructed to allow for several unique restriction sites during cloning and to enhance the positive-negative selection strategy by incorporating two HSV-tk genes. The pPNT based vector which has been used successfully by other labs has been modified to allow for easier linearization. Attempts to inactivate cathepsin L in ES cells are in progress.

组织蛋白酶原L是溶酶体半胱氨酸蛋白酶的前体 组织蛋白酶L 它是大量分泌的恶性 转化的小鼠细胞，以及用肿瘤促进剂或 生长因子 前组织蛋白酶L在恶性肿瘤中的功能不是 已知，但参与肿瘤侵袭和转移， 已经提出抑制对肿瘤的免疫应答。 在 在正常组织中，组织蛋白酶原L由肝脏，破骨细胞， 和支持细胞，表明可能参与骨吸收 和精子成熟。 为了研究 我们正试图通过以下方法来克隆这个基因： 胚胎干细胞（ES细胞）的插入诱变， 建立缺乏组织蛋白酶L功能的转基因小鼠。 我们有 克隆组织蛋白酶L DNA与ES细胞DNA同源， 载体（pPNT（s）和pSSC 9），其携带不同的启动子， 不同数量的单纯疱疹病毒（HSV）胸苷激酶（tk） 基因. 构建pSSC 9载体以允许几种独特的表达。 限制性酶切位点，并提高阳性-阴性 通过整合两个HSV-tk基因的选择策略。 基于PNT 已被其他实验室成功使用的载体， 修改为更容易线性化。 尝试将 组织蛋白酶L在胚胎干细胞中的作用。