Cre/LoxP-mediated inactivation of the murine Pten tumor suppressor gene
Cre/LoxP-mediated inactivation of the murine Pten tumor suppressor gene
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作者：
Lesche, R;Groszer, M;Wu, H
PTEN（或MMAC 1/TEP 1）肿瘤抑制基因在多种人类癌症和三种癌症易感综合征中频繁突变（Eng和Mackaocke，1998; Dahia，2000）。PTEN通过使磷脂酰肌醇3-激酶（PI 3激酶）的产物PIP 3去磷酸化来负调节PI 3信号传导途径（综述参见Cantley和Neel，1999）。在小鼠模型中Pten（染色体19）的失活证实了PTEN是真正的肿瘤抑制因子（Di Cristofano等人，1998; Podsypanina等人，1998; Suzuki等人，1998; Lesche等人，提交）：Pten+/J小鼠在多个器官中发生肿瘤，PtenJ/J小鼠在妊娠中期前的胚胎发生期间死亡。为了克服PtenJ/J小鼠的早期致死表型，并研究PTEN在胚胎发育、成年组织功能和肿瘤发生中的作用，我们已经产生了条件性Pten敲除小鼠品系。如图1所示，将LoxP序列插入外显子5侧翼的内源Pten基因座中。外显子5编码PTEN的磷酸酶结构域，其中已检测到许多肿瘤相关突变。将PtenloxP/+ ES细胞注射到C57/B6或Balb/c囊胚中。将嵌合小鼠与C57/B6或Balb/c小鼠回交，并通过Southern印迹和PCR基因分型确认PtenloxP/+等位基因的种系传递（未显示）。与PtenJ/J小鼠中观察到的胚胎致死表型相反，PtenloxP/loxP动物是存活的。在PtenloxP/loxP MEF细胞中检测到正常的PTEN水平和功能，并且长达两年没有观察到自发的肿瘤形成，这表明将loxP位点引入Pten基因座不会扰乱PTEN的正常功能。为了证明Cre介导的外显子5缺失，我们将PtenloXP/loXP动物与GFAP-Cre转基因小鼠杂交（Zhuo等人，2001年）旨在脑特异性删除。如图2c所示，PtenloxP/+; GFAP-Cre+/J小鼠中的Cre表达导致外显子5的神经特异性切除（泳道1-5）。相反，在其他非神经组织中可以检测到非常低的切除或没有切除（泳道6-9）。最后，我们表明在条件性缺失组织中没有检测到PTEN蛋白，并且已知的下游信号分子AKT/PKB被过度磷酸化（图2c）。因此，所产生的PtenloxP/loxP小鼠系将为研究PTEN在动物发育和肿瘤发生中的功能提供有价值的。
PTEN (or MMAC1/TEP1) tumor suppressor gene is frequently mutated in a variety of human cancers and in three cancer predisposition syndromes (Eng and Peacocke, 1998; Dahia, 2000). PTEN negatively regulates the phosphatidylinositol 3-kinase (PI3 kinase) signaling pathway by dephosphorylating PIP3, the product of PI3 kinase (for review, see Cantley and Neel, 1999). Inactivation of Pten (chromosome 19) in mouse models confirmed PTEN to be a bona fide tumor suppressor (Di Cristofano et al., 1998; Podsypanina et al., 1998; Suzuki et al., 1998; Lesche et al., submitted): Pten+/J mice developed tumors in multiple organs and PtenJ/J mice died during embryogenesis before midgestation. To overcome the early lethal phenotype in PtenJ/J mice and to study the roles of PTEN in embryonic development, adult tissue function, and tumorigenesis, we have generated a conditional Pten knockout mouse strain. LoxP sequences were inserted into the endogenous Pten locus flanking exon 5 as illustrated in Figure 1. Exon 5 encodes the phosphatase domain of PTEN in which many tumor-associated mutations have been detected. PtenloxP/+ ES cells were injected into either C57/B6 or Balb/c blastocysts. Chimeric mice were backcrossed to either C57/B6 or Balb/c mice and germ-line transmission of the PtenloxP/+ allele was confirmed by Southern blot and PCR genotyping (not shown). In contrast to the embryonic lethal phenotype observed in PtenJ/J mice, PtenloxP/loxP animals were viable. Normal PTEN level and function were detected in PtenloxP/loxP MEF cells and no spontaneous tumor formations were observed up to two years, suggesting that introducing the loxP sites into the Pten locus does not perturb the normal function of PTEN. To demonstrate Cre-mediated exon 5 deletion, we crossed PtenloxP/loxP animals with the GFAP-Cre transgenic mice (Zhuo et al., 2001) aimed for brain-specific deletion. As shown in Figure 2c, Cre expression in the PtenloxP/+; GFAP-Cre+/J mice resulted in neural-specific excision of exon 5 (lanes 1–5). In contrast, very low or no excision could be detected in other nonneural tissues (lanes 6–9). Finally, we showed that no PTEN protein could be detected in conditional deleted tissue and the known downstream signaling molecule AKT/PKB was hyperphosphorylated (Fig. 2c). Thus, the PtenloxP/loxP mouse line generated will be valuable for studying the function of PTEN in animal development and tumorigenesis.