In vivo electroporation-based conditional oncogenic activation implicates multiple distinct cellular origins for Group3 medulloblastoma. Mus musculus

We examined the transformation susceptibility of different cerebellar stem/progenitors by developing several new Group3 medulloblastoma murine models using orthotopic transplantation and in utero electroporation (EP)-based in vivo gene transfer with Cre/LoxP-mediated conditional Myc gene activation and loss of Trp53 function. We used microarrays to compared the transcriptome of these novel Group3 medulloblastoma mouse models and CPC mouse models to existing mouse models of medulloblastoma subgroups and used cross-species analysis to compare these models to human medulloblastoma subgroups Overall design: This study aimed to compare CPC models to other orthotopic models in GSE65888. Orthotopic cell-lineage specific MYC tumors were generated by enforced Myc expression in P6 GNPs isolated from P0-1 tamoxifen treated [Atoh1-CreER;Trp53fl/-] and [Prom1-CreER;Trp53fl/-] mice followed by cortical implants in immunocompromised mice. These tumors are referred to as Atoh1ER-MYC [dka072-075], Prom1-CreER [dka077-081]. The first Group3 MB models in which tumors developed in situ were generated by electroporation of plasmids containing Myc and dominant negative Trp53 flanked by loxP sites into the fourth ventricle of E13.5 Blbp-Cre [dka081, 087, 089, 090, 091 and blm121], Gad2-IRES-Cre [blm128-130 and blm134] and Ptf1a-Cre [blm135-137] mouse embryos. The gene expression profile of these tumors were compared to our previously published Group3 MB model as well as SHH and WNT models of medulloblastoma. For SHH subgroup medulloblastoma, [dka001-005, 009, 033 and 034] and [dka050-057], spontaneous medulloblastomas from [Cdkn2c-/-; Trp53Fl/Fl; Nestin-Cre] and [Cdkn2c-/-; Ptch1+/-] (Uziel et al.,2005 Genes Dev) were used, respectively. For Group3 medulloblastomas, [dka013-16, 049 and 065-067], in which Myc was overexpressed in Cdkn2c-/-, Trp53-/- cerebellar cells and implanted into the cortices of immunocompromised nude mice prior to tumor isolation. For WNT subgroup medulloblastomas [pgr003, 016 and 066], spontaneously developed tumors from CTNNB1+/lox (ex3); BLBP-Cre; Trp53Fl/Fl (Gibson et al., 2010, Nature) were removed for RNA extraction.

本研究通过两种策略构建多款新型第3组髓母细胞瘤（Group3 medulloblastoma）小鼠模型：一是采用原位移植技术，二是基于子宫内电穿孔（in utero electroporation, EP）的体内基因转染方法，结合Cre/LoxP介导的Myc条件性激活与Trp53功能缺失，以此探究不同小脑干细胞/祖细胞的转化易感性。我们通过微阵列技术，对比了这些新型第3组髓母细胞瘤小鼠模型与小脑祖细胞（cerebellar progenitor cells, CPC）小鼠模型的转录组（transcriptome），并结合已报道的不同亚型髓母细胞瘤小鼠模型开展分析；同时通过跨物种分析（cross-species analysis），将上述小鼠模型与人类髓母细胞瘤亚型进行比对。
实验设计：本研究旨在对比GSE65888数据集中的小脑祖细胞模型与其他原位移植模型。
原位细胞谱系特异性MYC肿瘤模型构建：从经他莫昔芬处理的P0-1龄[Atoh1-CreER;Trp53fl/-]与[Prom1-CreER;Trp53fl/-]小鼠中分离获得P6龄颗粒神经元前体细胞（granule neuron precursors, GNPs），强制过表达Myc后将细胞移植至免疫缺陷小鼠皮层，由此得到的肿瘤分别命名为Atoh1ER-MYC[dka072-075]与Prom1-CreER[dka077-081]。
首款原位生成肿瘤的第3组髓母细胞瘤模型，通过向E13.5龄的Blbp-Cre[dka081、087、089、090、091及blm121]、Gad2-IRES-Cre[blm128-130及blm134]与Ptf1a-Cre[blm135-137]小鼠胚胎的第四脑室，电穿孔转染携带Myc与loxP位点侧翼显性负向Trp53的质粒构建得到。
我们将上述肿瘤的基因表达谱，与本团队此前发表的第3组髓母细胞瘤模型，以及髓母细胞瘤的SHH（Sonic Hedgehog）亚型、WNT（Wnt信号通路）亚型模型进行对比。
针对SHH亚型髓母细胞瘤，分别使用[dka001-005、009、033及034]与[dka050-057]组样本，后者取自[Cdkn2c-/-;Trp53Fl/Fl;Nestin-Cre]与[Cdkn2c-/-;Ptch1+/-]小鼠的自发性髓母细胞瘤（Uziel等，2005，Genes Dev）。
针对第3组髓母细胞瘤，使用[dka013-16、049及065-067]组样本：该模型通过在Cdkn2c-/-、Trp53-/-小脑细胞中过表达Myc，随后将细胞移植至免疫缺陷裸小鼠皮层，待肿瘤形成后分离获取。
针对WNT亚型髓母细胞瘤，使用[pgr003、016及066]组样本：该组样本取自CTNNB1+/lox(ex3);BLBP-Cre;Trp53Fl/Fl小鼠自发形成的肿瘤（Gibson等，2010，Nature），用于RNA提取。