Cloning and expression of canine O6-methylguanine-DNA methyltransferase in target cells, using gammaretroviral and lentiviral vectors.
Cloning and expression of canine O6-methylguanine-DNA methyltransferase in target cells, using gammaretroviral and lentiviral vectors.
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使用γ逆转录病毒和慢病毒载体在靶细胞中克隆和表达犬O6-甲基鸟嘌呤-DNA 甲基转移酶。
DOI：
10.1089/104303404322959533
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Schuening,Friedrich
中科院分区：
文献类型：
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作者：
Zaboikin,Michail;Srinivasakumar,Narasimhachar;Zaboikina,Tatiana;Schuening,Friedrich
人O 6-甲基鸟嘌呤-DNA甲基转移酶（MGMT）基因及其突变体已被用于1，3-双（2-氯乙基）-1-亚硝基脲转导的造血干细胞的体内选择 （BCNU）单独或与O 6-苄基鸟嘌呤（BG）联合使用。为了在狗身上进行类似的体内选择，而不引起免疫反应，我们克隆了狗 MGMT耐药基因。犬和人的MGMT编码区的比较表明，有约62%的氨基酸的同一性和78%的相似性之间的两个MGMT。狗的MGMT也更长， 九种氨基酸。人MGMT的140位脯氨酸和周围氨基酸在犬序列中高度保守。为了确定是否在位置处的脯氨酸残基的突变 将犬MGMT中的144转化为赖氨酸将为选择转导的细胞提供与编码人突变体、莫洛尼鼠白血病病毒和人免疫缺陷1型载体类似的优势。 产生相应的突变MGMT，并用于在培养的细胞中分别表达犬和人的MGMT。单独使用BCNU或BCNU与BG的耐药性测定表明，野生型BCNU 而突变的犬MGMT提供了与人MGMT对应物相当的对选择剂的抗性。
The humanO6-methylguanine-DNA methyltransferase (MGMT) gene and its mutants have been used forin vivoselection of transduced hematopoietic stem cells with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) alone or in combination withO6-benzylguanine (BG). To allow similarin vivoselection in dogs, without the risk of inducing an immune response, we have cloned the canine MGMT drug resistance gene. Comparison of canine and human MGMT-coding regions indicates that there is about 62% amino acid identity and 78% similarity between the two MGMTs. The canine MGMT is also longer, by nine amino acids. Proline at position 140 and the surrounding amino acids of the human MGMT are highly conserved in the canine sequence. To determine whether mutation of the proline residue at position 144 to lysine in the canine MGMT would provide a similar advantage for selection of transduced cells as the human mutant, Moloney murine leukemia virus and human immunodeficiency type 1 vectors encoding the corresponding mutant MGMT were created and used to express separately canine and human MGMTs in cultured cells. Drug resistance assays using BCNU alone or BCNU with BG demonstrated that the wild-type and mutant canine MGMTs provided resistance to the selection agents that was comparable to the human MGMT counterparts.