Adult kidney functions in mice lacking genes that are essential for kidney development

我们的目的是产生缺乏肾脏发育必需基因的条件突变小鼠，并分析这些小鼠在成年阶段的肾脏功能。由于Sall1在后肾间充质（肾祖细胞）和成人肾脏中表达，我们计划在Sall1位点培养携带CreER的小鼠，CreER是Cre重组酶和突变雌激素受体的融合蛋白。当这只小鼠与具有多种发育基因的floxed小鼠杂交，并用ER配体他莫昔芬（tamoxifen）刺激时，理论上我们可以在所需的时间阶段删除基因。我们在胚胎干细胞中通过同源重组成功地将CreER插入Sall1位点，产生嵌合小鼠，随后获得Sall1 CreER小鼠。为了检验该小鼠株基因切除的效率，将其与lacZ指示小鼠杂交，并用他莫昔芬刺激。然而，lacZ染色较弱，表明切除率低。我们获得了普遍表达CreER的ROSA26CreER小鼠，并与指示小鼠杂交，但lacZ染色仍然较弱。虽然我们的他莫昔芬治疗方案可能不是最佳的，但很可能是CreER活性不够高，无法有效地删除基因，因为通过lacZ染色评估，在Sall1位点表达未修饰的Cre或无处不在的小鼠显示出强大的基因切除活性。因此，CreER可能不适合产生条件敲除小鼠。为了达到最初的目的，即分析成年阶段的肾功能，我们现在正在培养部分恢复Sall1活性的小鼠。

Our purpose was to generate conditional mutant mice lacking genes that are essential for kidney development, and to analyze renal functions of these mice at an adult stage. As Sall1 is expressed in the metanephric mesenchyme (renal progenitors) and in the adult kidney, we planned to generate mice harboring CreER, which is a fusion protein of Cre recombinase and a mutated estrogen receptor, in the Sall1 locus. When this mouse is crossed with floxed mice of a variety of developmental genes and is stimulated with tamoxifen, a ligand for ER, we could theoretically delete the genes at desired temporal stages. We succeeded in inserting CreER in the Sall1 locus by homologous recombination in embryonic stem cells, generated chimeric mice, and subsequently obtained Sall1 CreER mouse. To test the efficiency of gene excision using this mouse strain, it was crossed with lacZ indicator mouse and was stimulated with tamoxifen. The lacZ staining, however, was faint, indicating the low rate of excision. We then obtained ROSA26CreER mouse which expresses CreER ubiquitously, and crossed with the indicator mouse, but the lacZ staining was still weak. Though it is possible that our protocol of tamoxifen treatment was not optimal, it is likely that the CreER activity was not high enough to efficiently delete genes, because mice expressing unmodified Cre in the Sall1 locus or ubiquitously showed potent gene excision activity as assessed by the lacZ staining. Thus CreER may not be suitable to generate conditional knockout mice. To achieve the initial purpose that is the analysis of renal functions at the adult stage, we are now generating mice in which Sall1 activity is partially restored.