Jäger Richard, Maurer Jochen, Jacob Andrea, Schorle Hubert
Institute for Pathology, Department of Developmental Pathology, University of Bonn Medical School, Bonn, Germany.
Genesis. 2004 Mar;38(3):145-50. doi: 10.1002/gene.20014.
Development of inducible genetic switches for in vivo use with transgenic mice has revolutionized many areas in modern molecular biology. Combining two techniques, Cre/loxP-based genetic recombination and ligand-dependent activation of a chimeric protein, we generated transgenic mice which allow for the spatiotemporal control of expression and of activity of the proto-oncogene c-myc. To these ends, the gene encoding the tamoxifen-inducible c-mycER(T) fusion protein (mycER(T)) was inserted in the ubiquitously active ROSA 26 gene locus by gene targeting. In the resulting ROSAMER allele, generalized transcription of the mycER(T) gene is prevented by a preceding transcriptional stop sequence which is flanked by loxP sites. Crosses of ROSAMER transgenic mice with Mox2 cre transgenic mice revealed tight control of mycER(T) transcription in various tissues unless the transcriptional stop sequence was removed by cre-mediated excision. Furthermore, we were able to demonstrate tamoxifen-dependent activation of the MycER(T) protein in embryonic fibroblasts derived from such mice. As a proof of principle, we demonstrate that primary neural crest cultures established from ROSAMER mice maintain their proliferative capacity in a 4-OHT-dependent manner. Furthermore, we demonstrate that such neural crest cells retain their differentiation potential as shown by expression of NF 160, a marker of neuronal differentiation upon 4-OHT withdrawal. The transgenic mice produced may thus be valuable tools for studying the cell type-specific effects of c-myc activity in development and disease.
用于转基因小鼠体内的可诱导基因开关的开发彻底改变了现代分子生物学的许多领域。结合两种技术，即基于Cre/loxP的基因重组和嵌合蛋白的配体依赖性激活，我们培育出了转基因小鼠，可对原癌基因c-myc的表达和活性进行时空控制。为此，通过基因打靶将编码他莫昔芬诱导型c-mycER(T)融合蛋白（mycER(T)）的基因插入到普遍活跃的ROSA 26基因位点。在产生的ROSAMER等位基因中，mycER(T)基因的普遍转录被一个位于loxP位点两侧的转录终止序列所阻止。将ROSAMER转基因小鼠与Mox2 cre转基因小鼠杂交发现，除非转录终止序列被cre介导的切除所去除，否则在各种组织中mycER(T)转录受到严格控制。此外，我们能够证明在源自此类小鼠的胚胎成纤维细胞中，MycER(T)蛋白具有他莫昔芬依赖性激活。作为原理验证，我们证明从ROSAMER小鼠建立的原代神经嵴培养物以4-羟基他莫昔芬依赖性方式维持其增殖能力。此外，我们证明此类神经嵴细胞保留了其分化潜能，如在4-羟基他莫昔芬撤除后神经元分化标志物NF 160的表达所示。因此，所产生的转基因小鼠可能是研究c-myc活性在发育和疾病中的细胞类型特异性作用的有价值工具。
