Drug selection of mutant methylguanine methyltransferase from different oncoretroviral backbones results in multilineage hematopoietic transgene expression in primary and secondary recipients.
Drug selection of mutant methylguanine methyltransferase from different oncoretroviral backbones results in multilineage hematopoietic transgene expression in primary and secondary recipients.
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来自不同肿瘤逆转录病毒骨架的突变型甲基鸟嘌呤甲基转移酶的药物选择导致初级和次级受体中的多谱系造血转基因表达。
DOI：
10.1089/152581603322286015
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发表时间：
2003
期刊：
影响因子：
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通讯作者：
Gerson,StantonL
中科院分区：
文献类型：
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作者：
Davis,BrianM;Reese,JaneS;Lingas,Karen;Gerson,StantonL
优化的造血基因治疗需要在所需靶细胞群中具有强表达的载体和选择表达转导细胞的能力。在耐药性的背景下， 为了在小鼠中选择再生造血干细胞，我们检测了药物选择基因G156 A突变O 6-甲基鸟嘌呤-DNA转导骨髓移植后的组织表达 甲基转移酶（G156 AMGMT）。为了获得更多关于选择对组织特异性基因表达的影响的严格性的经验，我们还询问了三种基因之间是否存在表达差异。 不同的致癌逆转录病毒骨架- MPSV、SF和MFG。比较O 6-苄基鸟嘌呤（BG）和1，3-双（2-氯乙基）-1-亚硝基脲（BCNU）药物筛选后MGMT的表达 in vivo.在小鼠移植用表达G156 A MGMT的MPSV、MFG或SF逆转录病毒载体转导的细胞并经药物处理后，观察到几乎完全被转导的祖细胞取代 在骨髓里。每个骨架支持体内所有四种造血谱系中的MGMT表达，表明MGMT介导的选择确实是稳健的。在骨髓、脾脏和胸腺中的表达非常高， 载体之间的相似性和差异最可能是由于每个选定细胞的基因拷贝数的差异。在初次和二次接受者中，MFG和 这是以每个细胞最大的前病毒拷贝数转导的载体。这些数据表明，使用MGMT基因来保护初次和二次再繁殖鼠的强选择压力， BCNU的毒性。无论使用何种载体骨架，均观察到多器官表达，而无基因沉默的证据。这些数据有助于建立突变的BG抗性MGMT， 一种用于体内干细胞选择的有效选择基因。
Optimized hematopoietic gene therapy requires vectors with strong expression in the desired target cell population and the ability to select for the expressing transduced cells. In the context of drug resistance selection of repopulating hematopoietic stem cells in the mouse, we examined tissue expression after transduced marrow transplantation of the drug selection gene, G156A mutantO6-methylguanine-DNA methyltransferase (G156A MGMT). To gain more experience with the rigor of the impact of selection on tissue-specific gene expression, we also asked whether there are expression differences between three different onco-retroviral backbones - MPSV, SF, and MFG. MGMT expression was compared afterO6-benzylguanine (BG) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) drug selection in vivo. After mice were transplanted with cells transduced with MPSV, MFG, or SF retroviral vectors expressing G156A MGMT and drug treated, nearly complete replacement by transduced progenitors was observed in the marrow. Each backbone supported MGMT expression in all four hematopoietic lineages in vivo indicating that MGMT-mediated selection is indeed robust. Expression in marrow, spleen, and thymus was very similar between the vectors and differences were most likely due to differences in gene copy number per selected cell. In primary and secondary recipients, the highest expression was observed in MFG and this was the vector that transduced at the greatest proviral copy number per cell. These data indicate that strong selection pressure using the MGMT gene to protect primary and secondary repopulating murine stem cells from the toxicity of BCNU. Regardless of the vector backbone used, multiorgan expression was observed without evidence of gene silencing. These data help establish mutant, BG-resistant MGMT as a potent selection gene for stem cell selection in vivo.