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所必需的基因的条件性突变小鼠，并分析这些小鼠在成年阶段的肾功能。由于SALL1在后肾间充质(肾祖细胞)和成年肾脏中表达，我们计划在SALL1基因座上培育出CRE重组酶和突变的雌激素受体的融合蛋白Creer的小鼠。当这只小鼠与不同发育基因的小鼠杂交，并用ER的配体他莫昔芬刺激时，理论上我们可以在所需的时间阶段删除这些基因。我们通过胚胎干细胞中的同源重组，成功地将Creer插入到SALL1基因座上，并产生了嵌合小鼠，随后获得了SALL1 Creer小鼠。为了检验该品系小鼠的基因切除效率，将其与LacZ指示性小鼠杂交，并用他莫昔芬刺激。然而，Lacz染色很淡，表明切除率很低。然后我们获得了普遍表达Creer的ROSA26Creer小鼠，并与指示鼠杂交，但LacZ染色仍然很弱。尽管我们的三苯氧胺治疗方案可能不是最优的，但很可能CRER活性不够高，不足以有效删除基因，因为在SALL1基因座表达未经修饰的Cre的小鼠或通过LacZ染色评估的普遍存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性基因敲除小鼠。为了达到最初的目的，即分析成年阶段的肾功能，我们现在正在培育SALL1活性部分恢复的小鼠。

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.