Differential Gene Expression in Limbs of Wild Type and Lmx1b KO mice during limb dorsalization (e11.5, e12.5 & e13.5)

Lmx1b regulates dorsalization of limb fates, but the mechanism of this regulation has not been characterized. To identify candidate genes regulated by Lmx1b we compared the limbs from Lmx1b KO mice to wild type mice during limb dorsalization (e11.5-13.5). Differentially expressed genes that we common to all three stages examined were considered to be likely candidates for Lmx1b regulation and further evaluated. At 11.5 and 12.5 dpc, embryos were harvested and the limb buds with the limb girdles were isolated. Embryos at 13.5dpc were also harvested and their distal limb buds (zeugopods and autopods) were isolated. Embryos were genotyped to confirm Lmx1b homozygosity (-/- or +/+). RNA from embryonic forelimbs and hindlimbs of wild type (WT) and Lmx1b KO mice was harvested using the Rneasy Kit (Qiagen). RNA was pooled to decrease genetic variability, i.e., six limbs at 11.5 dpc, three limbs at 12.5 dpc and six limbs at 13.5 dpc. Duplicate samples were generated using different embryos for each stage and then hybridized to the Affymetrix GeneChip Mouse Genome 430 2.0 Array (UCI, Irvine, CA).Lmx1b可调控肢体命运的背侧化进程，但该调控的具体分子机制尚未阐明。为鉴定受Lmx1b调控的候选基因，本研究在肢体背侧化阶段（胚胎日龄e11.5至e13.5），对比了Lmx1b基因敲除（Knockout, KO）小鼠与野生型（Wild Type, WT）小鼠的肢体组织。在全部三个检测阶段均存在差异表达的基因被认定为Lmx1b调控的潜在候选基因，并开展进一步验证。在胚胎日龄11.5dpc与12.5dpc时，收集胚胎并分离带有肢带的肢芽组织；在13.5dpc的胚胎中，同样收集胚胎并分离其远端肢芽（中肢体节zeugopods与远肢体节autopods）。对胚胎进行基因型鉴定，以确认其Lmx1b基因的纯合状态（-/-或+/+）。使用Qiagen公司的RNeasy试剂盒（RNeasy Kit）提取野生型与Lmx1b基因敲除小鼠胚胎前肢与后肢的总RNA。为降低遗传异质性，对RNA样本进行混合：11.5dpc样本混合6个肢体的RNA，12.5dpc样本混合3个肢体的RNA，13.5dpc样本混合6个肢体的RNA。每个实验阶段均采用不同胚胎制备重复样本，随后将样本与Affymetrix GeneChip Mouse Genome 430 2.0表达芯片进行杂交，实验由加利福尼亚州欧文市的加州大学欧文分校UCI完成。