Dacquin Romain, Starbuck Michael, Schinke Thorsten, Karsenty Gérard
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA.
Dev Dyn. 2002 Jun;224(2):245-51. doi: 10.1002/dvdy.10100.
Cell- and time-specific gene inactivation should enhance our knowledge of bone biology. Implementation of this technique requires construction of transgenic mouse lines expressing Cre recombinase in osteoblasts, the bone forming cell. We tested several promoter fragments for their ability to drive efficient Cre expression in osteoblasts. In the first mouse transgenic line, the Cre gene was placed under the control of the 2.3-kb proximal fragment of the alpha1(I)-collagen promoter, which is expressed at high levels in osteoblasts throughout their differentiation. Transgenic mice expressing this transgene in bone were bred with the ROSA26 reporter (R26R) strain in which the ROSA26 locus is targeted with a conditional LacZ reporter cassette. In R26R mice, Cre expression and subsequent Cre-mediated recombination lead to expression of the LacZ reporter gene, an event that can be monitored by LacZ staining. LacZ staining was detected in virtually all osteoblasts of alpha1(I)-Cre;R26R mice indicating that homologous recombination occurred in these cells. No other cell type stained blue. In the second line studied, the 1.3-kb fragment of osteocalcin gene 2 (OG2) promoter, which is active in differentiated osteoblasts, was used to drive Cre expression. OG2-Cre mice expressed Cre specifically in bone. However, cross of OG2-Cre mice with R26R mice did not lead to any detectable LacZ staining in osteoblasts. Lastly, we tested a more active artificial promoter derived from the OG2 promoter. The artificial OG2-Cre transgene was expressed by reverse transcriptase-polymerase chain reaction in cartilage and bone samples. After cross of the artificial OG2-Cre mice with R26R mice, we detected a LacZ staining in articular chondrocytes but not in osteoblasts. Our data suggest that the only promoter able to drive Cre expression at a level sufficient to induce recombination in osteoblasts is the alpha1(I)-collagen promoter.
细胞特异性和时间特异性的基因失活应能增进我们对骨生物学的了解。实施这项技术需要构建在成骨细胞（即形成骨骼的细胞）中表达Cre重组酶的转基因小鼠品系。我们测试了几个启动子片段驱动成骨细胞中高效Cre表达的能力。在第一个小鼠转基因品系中，Cre基因置于α1(I)-胶原启动子2.3 kb近端片段的控制之下，该片段在成骨细胞整个分化过程中均高水平表达。在骨骼中表达该转基因的转基因小鼠与ROSA26报告基因（R26R）品系杂交，其中ROSA26位点用条件性LacZ报告基因盒进行靶向。在R26R小鼠中，Cre表达及随后的Cre介导的重组导致LacZ报告基因表达，这一事件可通过LacZ染色进行监测。在α1(I)-Cre;R26R小鼠几乎所有成骨细胞中均检测到LacZ染色，表明这些细胞中发生了同源重组。没有其他细胞类型染成蓝色。在研究的第二个品系中，骨钙素基因2（OG2）启动子的1.3 kb片段（在分化的成骨细胞中具有活性）用于驱动Cre表达。OG2-Cre小鼠在骨骼中特异性表达Cre。然而，OG2-Cre小鼠与R26R小鼠杂交并未在成骨细胞中导致任何可检测到的LacZ染色。最后，我们测试了一个源自OG2启动子的活性更高的人工启动子。通过逆转录聚合酶链反应在软骨和骨样本中检测到人工OG2-Cre转基因的表达。人工OG2-Cre小鼠与R26R小鼠杂交后，我们在关节软骨细胞中检测到LacZ染色，但在成骨细胞中未检测到。我们的数据表明，唯一能够驱动Cre表达至足以在成骨细胞中诱导重组水平的启动子是α1(I)-胶原启动子。
