Retroviral vector design for long-term expression in murine hematopoietic cells in vivo
Retroviral vector design for long-term expression in murine hematopoietic cells in vivo
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小鼠造血细胞体内长期表达的逆转录病毒载体设计
DOI：
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发表时间：
1994
期刊：
影响因子：
--
通讯作者：
S. Karlsson
中科院分区：
文献类型：
--
作者：
P. H. Correll;S. Colilla;S. Karlsson
构建了一系列含有不同启动子驱动的人葡萄糖脑苷酶(GC)基因的逆转录病毒载体，以探索哪种载体设计可以最有效地转导小鼠造血干细胞，并驱动所转移的基因在由转导的干细胞重组的小鼠造血细胞中的表达。最简单的载体LG，其GC基因由病毒LTR驱动，是感染HSCs的最有效的载体，在受体小鼠的造血组织中，平均病毒拷贝数为3拷贝/细胞。一般来说，含有任何额外启动子或增强子以驱动GC基因或可选择标记基因(Neo)表达的病毒载体具有较低的滴度和/或以较低的效率转导HSC。当使用人磷酸甘油酸(PGK)启动子驱动人GC cDNA时，这一点最为明显。尽管多次尝试获得高滴度的生产者克隆，但这种病毒始终产生低滴度，并随后在长期重组的小鼠中产生最低的前病毒拷贝数。只有病毒LTR和PGK启动子能够在长期重组小鼠的造血细胞中驱动显著水平的人GC RNA，而内部疱疹病毒TK或SV40即刻早期启动子产生的RNA水平要低得多。与病毒LTRS相反的方向插入内部转录单位对基因表达有不利影响。与LG载体相比，含有骨髓增殖性肉瘤病毒增强子的杂合LTR在骨髓来源的巨噬细胞中产生的RNA水平高于骨髓非贴壁细胞。驱动人GC基因表达的内部启动子的存在似乎对病毒LTR的表达水平没有不利影响。事实上，在存在内部TK或PGK启动子的情况下，LTR的表达增加，尽管存在较低的前病毒拷贝数。将第二个基因(Neo)插入到载体中对体内造血细胞的长期表达产生了负面影响；然而，这似乎完全是由于该载体的转导效率较低。总体而言，长期重组小鼠巨噬细胞中GC活性的最高水平是由LG载体产生的；然而，这些水平是可变的。
A series of retroviral vectors containing the human glucocerebrosidase (GC) cDNA driven by various promoters have been constructed in an attempt to discover which vector design can most efficiently transduce murine hematopoietic stem cells (HSCs) and drive expression of the transferred gene in hematopoietic cells of mice reconstituted with the transduced stem cells. The simplest vector, LG, in which the GC gene is driven by the viral LTR, was the most efficient vector at infecting HSCs, with an average viral copy number in hematopoietic tissues of 3 copies/cell in recipient mice. In general, the viral vectors that contained any additional promoters or enhancers to drive expression of either the GC gene or a selectable marker gene (Neo) had lower titers and/or transduced HSCs at a lower efficiency. This was seen most markedly when the human phosphoglycerate (PGK) promoter was used to drive the human GC cDNA. Despite repeated attempts to obtain a high titer producer clone, this virus consistently produced low titers and subsequently resulted in the lowest proviral copy numbers in long-term reconstituted mice. Only the viral LTR and PGK promoter were capable of driving significant levels of human GC RNA in hematopoietic cells of long-term reconstituted mice, with a much lower level of RNA generated by an internal herpes TK or SV40 immediate early promoter. Insertion of the internal transcription unit in the opposite orientation relative to the viral LTRs had a detrimental effect on gene expression. The levels of RNA generated by a hybrid LTR containing the myeloproliferative sarcoma virus enhancer were higher in bone marrow-derived macrophages than in nonadherent cells of the bone marrow when compared with the LG vector. The presence of an internal promoter to drive expression of the human GC cDNA did not seem to have a detrimental effect on expression levels from the viral LTR. In fact, in the presence of an internal TK or PGK promoter expression from the LTR was increased despite the presence of lower proviral copy numbers. Insertion of a second gene (Neo) into the vector had a negative impact on long-term expression in hematopoietic cells in vivo; however, this seems to be due solely to the lower transduction efficiency of this vector. Overall, the highest levels of GC activity in macrophages of long-term reconstituted mice were generated by the LG vector; however, these levels were variable.(ABSTRACT TRUNCATED AT 400 WORDS)
人葡萄糖脑苷脂酶 cDNA 的分子克隆和核苷酸序列。
DOI：
10.1073/pnas.82.21.7289
发表时间：
1985
影响因子：
11.1
作者：
Sorge,J;West,C;Westwood,B;Beutler,E
通讯作者：
Beutler,E
鼠造血细胞中人腺苷脱氨酶的逆转录病毒基因转移：选择标记序列对长期表达的影响。
影响因子：
20.3
作者：
Apperley,JF;Luskey,BD;Williams,DA
通讯作者：
Williams,DA
移植逆转录病毒感染的造血干细胞的小鼠体内人腺苷脱氨酶的长期表达。
DOI：
10.1073/pnas.86.22.8892
发表时间：
1989
影响因子：
11.1
作者：
Lim,B;Apperley,JF;Orkin,SH;Williams,DA
通讯作者：
Williams,DA