Center for Neurodegeneration and Experimental Therapeutics, Department of Neurology, Heersink School of Medicine, University of Alabama at Birmingham, 1719 Sixth Avenue South, Civitan International Research Building 510A, Birmingham, AL, 35294, USA.
Department of Genetics, Heersink School of Medicine, University of Alabama at Birmingham, Birmingham, AL, 35294, USA.
Sci Rep. 2022 Jun 14;12(1):9837. doi: 10.1038/s41598-022-13557-8.
We report an incidental 358.5 kb deletion spanning the region encoding for alpha-synuclein (αsyn) and multimerin1 (Mmrn1) in the Rab27a/Rab27b double knockout (DKO) mouse line previously developed by Tolmachova and colleagues in 2007. Western blot and RT-PCR studies revealed lack of αsyn expression at either the mRNA or protein level in Rab27a/b DKO mice. PCR of genomic DNA from Rab27a/b DKO mice demonstrated at least partial deletion of the Snca locus using primers targeted to exon 4 and exon 6. Most genes located in proximity to the Snca locus, including Atoh1, Atoh2, Gm5570, Gm4410, Gm43894, and Grid2, were shown not to be deleted by PCR except for Mmrn1. Using whole genomic sequencing, the complete deletion was mapped to chromosome 6 (60,678,870-61,037,354), a slightly smaller deletion region than that previously reported in the C57BL/6J substrain maintained by Envigo. Electron microscopy of cortex from these mice demonstrates abnormally enlarged synaptic terminals with reduced synaptic vesicle density, suggesting potential interplay between Rab27 isoforms and αsyn, which are all highly expressed at the synaptic terminal. Given this deletion involving several genes, the Rab27a/b DKO mouse line should be used with caution or with appropriate back-crossing to other C57BL/6J mouse substrain lines without this deletion.
我们报道了一个偶然发现的 358.5kb 缺失，跨越了编码α-突触核蛋白（αsyn）和多聚蛋白 1（Mmrn1）的区域，该缺失发生在 2007 年由 Tolmachova 及其同事先前开发的 Rab27a/Rab27b 双敲除（DKO）小鼠品系中。Western blot 和 RT-PCR 研究显示，Rab27a/b DKO 小鼠中 αsyn 的 mRNA 或蛋白水平均无表达。使用针对外显子 4 和外显子 6 的引物对 Rab27a/b DKO 小鼠的基因组 DNA 进行 PCR 分析，证实了 Snca 基因座至少部分缺失。除了 Mmrn1 之外，靠近 Snca 基因座的大多数基因，包括 Atoh1、Atoh2、Gm5570、Gm4410、Gm43894 和 Grid2，都没有被 PCR 删除。通过全基因组测序，将完全缺失区域定位到染色体 6（60,678,870-61,037,354），该缺失区域比以前在 Envigo 维持的 C57BL/6J 亚系中报道的要小一些。这些小鼠皮层的电子显微镜显示，突触末端异常增大，突触小泡密度降低，提示 Rab27 同工型和 αsyn 之间可能存在相互作用，因为它们在突触末端都高度表达。鉴于这种缺失涉及多个基因，因此应谨慎使用 Rab27a/b DKO 小鼠品系，或在与不发生这种缺失的其他 C57BL/6J 小鼠亚系进行适当回交后再使用。
