Vinh D B, McIvor R S
Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis.
J Pharmacol Exp Ther. 1993 Nov;267(2):989-96.
To determine the effect of in vivo pharmacological selective pressure on the insertion and expression of new gene sequences, retroviral-mediated transfer of a methotrexate-resistant dihydrofolate reductase (Mtxr-DHFR) gene in hematopoietic tissue was investigated using a murine syngeneic bone marrow transplant system. A series of recombinant retroviral vectors were constructed to contain long terminal repeat (LTR) regions from different murine retroviruses associated with various proliferative disorders of the lymphohematopoietic system, including Moloney leukemia virus, spleen focus-forming virus (anemia strain) and myeloproliferative sarcoma virus. High-titer DHFR virus (10(7) colony-forming units/ml) was generated by gene amplification adapting virus-producer cell lines to grow in medium containing increasing concentrations of Mtx. This high-titer DHFR virus was used to introduce the Mtxr-DHFR gene into murine hematopoietic tissue by injecting DHFR virus-exposed marrow into lethally irradiated syngeneic recipient mice that subsequently were administered Mtx. Southern blot analysis of spleen DNA demonstrated insertion of the DHFR provirus in all surviving mice transplanted with DHFR virus-exposed marrow. However, enzymatic assay of crude spleen extracts demonstrated the presence of Mtxr-DHFR activity only in mice that were administered Mtx; nonadministered animals or animals transplanted with control (neo) virus-infected marrow contained undetectable drug-resistant enzyme activity. These results suggest the selective outgrowth of hematopoietic tissue harboring and expressing a DHFR provirus in animals administered Mtx and have implications for the application of drug-resistance gene insertion in somatic tissues of animals and humans.
为了确定体内药理学选择性压力对新基因序列插入和表达的影响，利用小鼠同基因骨髓移植系统研究了逆转录病毒介导的甲氨蝶呤抗性二氢叶酸还原酶（Mtxr-DHFR）基因在造血组织中的转移。构建了一系列重组逆转录病毒载体，使其包含来自与淋巴造血系统各种增殖性疾病相关的不同小鼠逆转录病毒的长末端重复序列（LTR）区域，包括莫洛尼白血病病毒、脾集落形成病毒（贫血株）和骨髓增殖性肉瘤病毒。通过使病毒生产细胞系在含有浓度递增的甲氨蝶呤的培养基中生长进行基因扩增，产生了高滴度的DHFR病毒（10⁷集落形成单位/毫升）。将这种高滴度的DHFR病毒通过将暴露于DHFR病毒的骨髓注射到经致死性照射的同基因受体小鼠体内，从而将Mtxr-DHFR基因引入小鼠造血组织，随后给这些小鼠施用甲氨蝶呤。对脾脏DNA进行的Southern印迹分析表明，在所有移植了暴露于DHFR病毒的骨髓的存活小鼠中，DHFR前病毒均有插入。然而，对脾脏粗提物进行的酶活性测定表明，仅在施用了甲氨蝶呤的小鼠中存在Mtxr-DHFR活性；未施用甲氨蝶呤的动物或移植了对照（neo）病毒感染骨髓的动物含有无法检测到的耐药酶活性。这些结果表明，在施用甲氨蝶呤的动物中，含有并表达DHFR前病毒的造血组织有选择性的生长，这对在动物和人类体细胞组织中插入耐药基因的应用具有启示意义。
