Comparison of small RNA profiling in sperm susceptible or not susceptible to epigenetic silencing of the LoxP site

During our attempt to engineer a conditional Kdm1a knockout using germline expressed Vasa-Cre transgene, we observed that the efficiency of LoxP recombination is dramatically reduced after the first recombination event occurs. This is due to a transvection event in which the recombination of one allele in one generation, blocks the recombination of the other allele in the following generation. Interestingly, this inhibition can be relieved by introducing an additional cross to a wildtype mouse between the first and the second Kdm1a recombination event. The recombination inhibition is therefore the result of epigenetic silencing that is erased once spermatogenesis is allowed to happen in the absence of Vasa-Cre. After excluding other possibilities, we considered the hypothesis that a diffusible molecule targeting the LoxP site that could be deposited in sperm following the first Cre recombination event (original cross). This molecule should not be present anymore when the sperm is collected after the additional cross to wildtype (extra cross). To test whether this diffusible molecule could be a small RNA, we produced and compared small RNA libraries prepared from sperm derived from the original cross and from the extra cross. These samples are genetically identical but differ in their parental history as well as in the ability to recombine the second floxed Kdm1a allele in the following generation. The comparison aimed at finding potential small RNAs mapping to or close to the loxP site present only, or more abundantly, in the original cross. Unfortunately, we were not able to identify such small RNAs and we also could not find any consistent difference in small RNA profiles among the different samples. We therefore concluded that the transvection event is not connected to a diffusible small RNA accumulated in sperm. Sperm was isolated from the cauda epidydimis and washed with PBS. Trizol was used to extract RNA from the sperm samples with slight modifications retain small RNAs. Sperm from each mouse was processed separately and treated as a replicate. Two biological replicates per condition were produced. For each sample, 250-1500ng of RNA were used for size-selection on a polyacrylamide gel. After elution from the gel we proceeded to library preparation.本研究在尝试利用生殖系表达的Vasa-Cre转基因（Vasa-Cre transgene）构建条件性Kdm1a基因敲除模型时，观察到首次LoxP位点重组（LoxP recombination）发生后，后续LoxP重组效率显著降低。该现象源于一种反式互作事件：某一代中单个等位基因的重组，会抑制下一代中另一等位基因的重组。有趣的是，在首次与第二次Kdm1a基因重组事件之间引入一次与野生型小鼠的回交，即可解除这种抑制作用。因此，这种重组抑制现象实为表观遗传沉默（epigenetic silencing）所致，且当在无Vasa-Cre的条件下允许精子发生完成后，该表观沉默会被消除。在排除其他潜在可能性后，我们提出如下假说：首次Cre重组事件（原始回交）后，精子中会沉积一种靶向LoxP位点的可扩散分子；而在额外回交至野生型小鼠后收集的精子中，该分子将不再存在。为验证该可扩散分子是否为小RNA，我们分别构建并比较了源自原始回交与额外回交小鼠精子的小RNA文库（small RNA libraries）。这些样本的遗传背景完全一致，但亲本来源历史不同，且下一代中第二等位基因的floxed Kdm1a等位基因（floxed Kdm1a allele）重组能力也存在差异。本比对旨在筛选仅在原始回交样本中存在、或在其中丰度更高的、靶向LoxP位点或其邻近区域的潜在小RNA。遗憾的是，我们未能鉴定到符合上述特征的小RNA，且不同样本间的小RNA表达谱也未发现一致的差异。据此我们得出结论：该反式互作事件与精子中积累的可扩散小RNA并无关联。本研究从附睾尾（cauda epidydimis）分离精子，并用磷酸盐缓冲液（PBS）洗涤。采用经轻微改良的Trizol试剂（Trizol）法提取精子样本中的RNA，以保留小RNA组分。每只小鼠的精子单独处理，作为一个生物学重复；每个条件设置2个生物学重复。每个样本取250~1500ng RNA用于聚丙烯酰胺凝胶（polyacrylamide gel）电泳进行片段大小筛选。从凝胶中洗脱RNA后，即开展文库制备工作。