SYNTHESIS AND FUNCTION OF A TRANSFORMATION-DEPENDENT SECRETED LYSOSOMAL PROTEASE

组织蛋白酶L是溶酶体半胱氨酸蛋白酶的前体 组织蛋白酶L是由恶性黑色素瘤大量分泌。 转化的小鼠细胞，以及用肿瘤促进剂或 增长因素。原组织蛋白酶L在恶性肿瘤中的作用 已知，但参与肿瘤侵袭和转移以及 抑制对肿瘤的免疫反应已被提出。在……里面 在正常组织中，L原蛋白是由肝脏、破骨细胞、 和支持细胞，提示可能参与骨吸收 和精子成熟。研究其生物学功能 在L的研究中，我们正试图通过以下方法使该基因失活 在胚胎干细胞中插入诱变的目的 建立组织蛋白酶L功能缺失的转基因小鼠。我们有 克隆组织蛋白酶L同源基因与ES细胞DNA成双靶点 载体(pPNT(S)和pSSC9)，携带不同的启动子和 不同数量的单纯疱疹病毒胸苷激酶(Tk) 基因。PSSC9载体被构建为允许几个唯一 克隆过程中的限制性酶切位点和增强的阳性-阴性 整合两个HSV-tk基因的选择策略。基于PPNT的 已经被其他实验室成功使用的载体已经被 对其进行了修改，以便更容易线性化。尝试停用 组织蛋白酶L在ES细胞中的作用正在进行中。

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.