Expression of human adenosine deaminase in murine haematopoietic progenitor cells following retroviral transfer
Expression of human adenosine deaminase in murine haematopoietic progenitor cells following retroviral transfer
复制标题
逆转录病毒转移后人腺苷脱氨酶在小鼠造血祖细胞中的表达
作者：
J. Belmont;J. Henkel;Stephen M. W. Chang;K. Wager;R. Kellems;J. Dick;M. Magli;R. A. Phillips;A. Bernstein;C. Caskey
腺苷脱氨酶（ADA）缺乏症是一种常染色体隐性遗传性代谢缺陷，可导致男性严重的联合免疫缺陷1。这种酶，虽然在大多数组织中组成型表达，但在未成熟的T细胞中以高水平表达，并且对该疾病的病理生理学的研究表明，增加的脱氧腺苷或改变的甲基化能力对T细胞成熟具有毒性作用1。虽然骨髓移植可以纠正免疫缺陷2，3，这种疗法与移植物抗宿主病和不完全的免疫恢复有关，因此我们的实验室和其他人试图开发一种基因替代的方法，作为治疗这种疾病的可能方法4。此外，人ADA基因及其一些突变体的互补DNA的表征5，6使得设计基因转移策略成为可能。我们现在已经将人腺苷脱氨酶cDNA亚克隆到逆转录病毒穿梭载体pZIP-SV（B）中，并以这种方式分离出一种细胞系4.2T，该细胞系产生高滴度的复制缺陷型逆转录病毒，该病毒已被用于将人ADA基因转移到小鼠骨髓细胞中。用体外集落形成实验证明，在抗生素G418或9-呋喃木糖苷加脱氧共形霉素的作用下，新霉素抗性基因（neo）和ADA基因在小鼠骨髓集落形成单位（CFU）中转移和表达，同工酶分析也表明人ADA在培养的小鼠骨髓中表达。
Adenosine deaminase (ADA) deficiency, an autosomal recessive inborn error of metabolism, leads to severe combined immune deficiency in man1. This enzyme, although constitutively expressed in most tissues, is expressed at high level in immature T cells, and study of the pathophysiology of the disorder indicates that increased deoxyadenosine or altered methylation capacity have toxic effects on T-cell maturation1. Although bone marrow transplantation can correct the immune deficiency2,3, this therapy is associated with graft-versus-host disease and incomplete immune restoration,and so our laboratory and others have sought to develop a method of gene replacement as a possible treatment for the disease4. Moreover, characterization of the complementary DNA of the human ADA gene and some of its mutants5,6 makes it possible to design gene transfer strategies. We have now sub-cloned a human adenosine deaminase cDNA into the retrovirus shuttle vector pZIP-SV(B),and in this way have isolated a cell line, 4.2T, which produces high titres of replication-defective retro-virus which have been used to transfer the gene for human ADA to mouse bone marrow cells. Transfer and expression of the neomycin-resistance gene (neo) and the ADA gene in murine bone marrow colony-forming units (CFU) was demonstrated by in vitro colony formation in the presence of the antibiotic G418 or 9-xylofuranosyladenine plus deoxycoformycin, respectively.Isoenzyme analysis also showed human ADA expression in the cultured mouse bone marrow.
在培养的小鼠细胞中选择性过量产生腺苷脱氨酶。
DOI：
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发表时间：
1983
期刊：
The Journal of biological chemistry
影响因子：
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作者：
Yeung,CY;Ingolia,DE;Bobonis,C;Dunbar,BS;Riser,ME;Siciliano,MJ;Kellems,RE
通讯作者：
Kellems,RE