Lichtenheld M G, Podack E R, Levy R B
Department of Microbiology and Immunology, University of Miami, School of Medicine, FL 33101.
J Immunol. 1995 Mar 1;154(5):2153-63.
Perforin is a pore-forming effector molecule of CTL and NK cells. To characterize perforin gene expression and its transcriptional control mechanisms in vivo, expression of a cell surface tag, i.e., human CD4, was driven by 5.1 kb of the murine perforin 5' flanking and promoter region in transgenic mice. Six out of seven transgenic lines expressed the perforin-tag hybrid gene at low to intermediate levels, depending on the integration site. Tissues not yet reported to contain perforin-expressing lymphocytes were identified. Transgene expression occurred in all cells that physiologically are able to express perforin, i.e., in T cells and NK cells, and in some T cells that normally may express little or no perforin. At the whole organ level, significant amounts of transgenic mRNA and endogenous perforin mRNA were co-expressed in the lymphoid organs, as well as in the lung, the ileum, the oviduct/uterus, and the bone marrow. At the single cell level, the perforin tag was present on NK cells and on CD8+, as well as on CD4+ T cells. Also targeted were Thy-1.2+ gamma delta T cells, but not Thy1.2- gamma delta T cells, B cells, nor monocytes. During thymic T cell development, transgene expression occurred in double negative (CD4-CD8-) thymocytes and was detected at all subsequent stages, but exceeded the expression levels of the endogenous gene in the thymus. In conclusion, the analyzed perforin 5' flanking and promoter region contains important cis-acting sequences that restrict perforin expression to T cells and NK cells, and therefore provides a unique tool for manipulating T cell and/or NK cell-mediated immune responses in transgenic mice. On the other hand, the normal control of perforin gene expression involves at least one additional negative control mechanism that was not mediated by the transgenic promoter and upstream region. This control restricts perforin gene expression in thymically developing T cells and in most resting peripheral T cells, but can be released upon T cell activation.
穿孔素是细胞毒性T淋巴细胞（CTL）和自然杀伤细胞（NK细胞）的一种成孔效应分子。为了在体内表征穿孔素基因表达及其转录调控机制，在转基因小鼠中，通过5.1 kb的小鼠穿孔素5'侧翼和启动子区域驱动细胞表面标签即人CD4的表达。七个转基因品系中有六个以低到中等水平表达穿孔素-标签杂交基因，这取决于整合位点。发现了尚未报道含有表达穿孔素淋巴细胞的组织。转基因表达发生在所有生理上能够表达穿孔素的细胞中，即T细胞和NK细胞，以及一些通常可能很少或不表达穿孔素的T细胞中。在整个器官水平上，大量转基因mRNA和内源性穿孔素mRNA在淋巴器官以及肺、回肠、输卵管/子宫和骨髓中共同表达。在单细胞水平上，穿孔素标签存在于NK细胞、CD8 +以及CD4 + T细胞上。Thy-1.2 + γδT细胞也有靶向表达，但Thy1.2 - γδT细胞、B细胞和单核细胞没有。在胸腺T细胞发育过程中，转基因表达发生在双阴性（CD4 - CD8 -）胸腺细胞中，并在所有后续阶段都能检测到，但在胸腺中超过了内源性基因的表达水平。总之，分析的穿孔素5'侧翼和启动子区域包含重要的顺式作用序列，这些序列将穿孔素表达限制在T细胞和NK细胞中，因此为在转基因小鼠中操纵T细胞和/或NK细胞介导的免疫反应提供了独特的工具。另一方面，穿孔素基因表达的正常调控涉及至少一种额外的负调控机制，该机制不是由转基因启动子和上游区域介导的。这种调控限制了胸腺发育中的T细胞和大多数静止外周T细胞中的穿孔素基因表达，但在T细胞激活后可以解除。
