PDF(Pubmed)
Abstract：
The Cre-lox system is an indispensable tool in neuroscience research for targeting gene deletions to specific cellular populations. Here we assess the utility of several transgenic Cre lines, along with a viral approach, for targeting cerebellar Purkinje cells (PCs) in mice. Using a combination of a fluorescent reporter line (Ai14) to indicate Cre-mediated recombination and a floxed Dystroglycan line (Dag1flox ), we show that reporter expression does not always align precisely with loss of protein. The commonly used Pcp2Cre line exhibits a gradual mosaic pattern of Cre recombination in PCs from Postnatal Day 7 (P7) to P14, while loss of Dag1 protein is not complete until P30. Ptf1aCre drives recombination in precursor cells that give rise to GABAergic neurons in the embryonic cerebellum, including PCs and molecular layer interneurons. However, due to its transient expression in precursors, Ptf1aCre results in stochastic loss of Dag1 protein in these neurons. NestinCre , which is often described as a \"pan-neuronal\" Cre line for the central nervous system, does not drive Cre-mediated recombination in PCs. We identify a Calb1Cre line that drives efficient and complete recombination in embryonic PCs, resulting in loss of Dag1 protein before the period of synaptogenesis. AAV8-mediated delivery of Cre at P0 results in gradual transduction of PCs during the second postnatal week, with loss of Dag1 protein not reaching appreciable levels until P35. These results characterize several tools for targeting conditional deletions in cerebellar PCs at different developmental stages and illustrate the importance of validating the loss of protein following recombination.
摘要：
Cre-lox系统是神经科学研究中将基因缺失靶向特定细胞群体的不可或缺的工具。在这里，我们评估了几种转基因Cre系的实用性，随着病毒的方法，用于靶向小鼠小脑浦肯野细胞。使用荧光报道系（Ai14）的组合来指示Cre介导的重组和floxedDystroglycan系（Dag1flox），我们表明，报道分子表达并不总是与蛋白质丢失精确对齐。常用的Pcp2Cre系在P7-P14的Purkinje细胞中表现出Cre重组的逐渐镶嵌模式，而Dag1蛋白的丢失直到P30才完全。Ptf1aCre驱动前体细胞的重组，从而在胚胎小脑中产生GABA能神经元，包括浦肯野细胞和分子层中间神经元。然而,由于它在前体中的瞬时表达，Ptf1aCre导致这些神经元中Dag1蛋白的随机丢失。NestinCre,通常被描述为中枢神经系统的“泛神经元”Cre线，在浦肯野细胞中不驱动Cre介导的重组。我们确定了Calb1Cre系，它可以在胚胎Purkinje细胞中驱动有效和完全的重组，导致Dag1蛋白在突触发生之前丢失。在P0时AAV8介导的Cre递送导致在出生后第二周期间Purkinje细胞的逐渐转导，Dag1蛋白的损失直到P35才达到可感知的水平。这些结果表征了在不同发育阶段靶向小脑浦肯野细胞中条件性缺失的几种工具，并说明了验证重组后蛋白质丢失的重要性。重要性陈述用于将基因缺失靶向定义的细胞群体的Cre系的发展导致了神经科学中的重要发现。和任何工具一样，有固有的局限性，必须仔细考虑。在这里，我们描述了几种Cre系，可用于在各个发育阶段靶向小脑Purkinje细胞。我们使用Cre依赖性荧光报道系和突触支架分子Dystroglycan的条件性缺失的组合作为示例，以突出显示荧光报道分子的存在与蛋白质丢失之间的潜在脱节。
