Holland C A, Anklesaria P, Sakakeeny M A, Greenberger J S
Department of Radiation Oncology, University of Massachusetts Medical Center, Worcester 01605.
Proc Natl Acad Sci U S A. 1987 Dec;84(23):8662-6. doi: 10.1073/pnas.84.23.8662.
To analyze the transcriptional activity of retroviral enhancer sequences in hematopoietic lineages, we determined the effect of enhancer sequences on the expression of the neomycin resistance gene transferred by two retroviral vectors to primary hematopoietic lineages. We constructed the vector pFr-SV(X). The Moloney murine leukemia virus enhancer region of a vector, pZIP-SV(X), was replaced by a 380-nucleotide-long fragment containing the enhancer sequences of the Friend murine leukemia virus. The enhancer sequences of Friend murine leukemia virus were used because these sequences have been shown to target the disease specificity of the virus to the erythroid lineage. Hematopoietic progenitors in murine continuous marrow cultures were infected with identical numbers of pure defective, infectious viral vector particles of either pFr-SV(X) or pZIP-SV(X). Expression of the transferred neomycin resistance gene in multipotential stem cells and their differentiated progeny was assayed as the ability of infected progenitors to form colonies (greater than 50 cells) in G418. Expression of the neomycin resistance gene in multipotential progenitor cells during the entire 11 weeks of the cultures was independent of the vector used to transfer the gene. Conversely, committed hemoglobinized erythroid bursts and myeloid colonies resistant to G418 were consistently produced by pFr-SV(X)-infected cultures but not pZIP-SV(X)-infected cultures. These results demonstrate that both pFr-SV(X) and pZIP-SV(X) were stably integrated and expressed in more primitive, multilineage, hematopoietic progenitor cells and suggest that the enhancer sequences of a vector affects expression of the transferred neomycin resistance gene when these cells differentiate to committed myeloid and erythroid cells.
为了分析逆转录病毒增强子序列在造血谱系中的转录活性，我们确定了增强子序列对两种逆转录病毒载体转移至原代造血谱系的新霉素抗性基因表达的影响。我们构建了载体pFr-SV(X)。将载体pZIP-SV(X)的莫洛尼鼠白血病病毒增强子区域替换为一个380个核苷酸长的片段，该片段包含弗氏鼠白血病病毒的增强子序列。使用弗氏鼠白血病病毒的增强子序列是因为这些序列已被证明可将病毒的疾病特异性靶向红系谱系。用相同数量的pFr-SV(X)或pZIP-SV(X)的纯缺陷型、感染性病毒载体颗粒感染小鼠连续骨髓培养物中的造血祖细胞。将感染的祖细胞在G418中形成集落（大于50个细胞）的能力作为多能干细胞及其分化后代中转移的新霉素抗性基因的表达进行测定。在整个11周的培养过程中，多能祖细胞中新霉素抗性基因的表达与用于转移该基因的载体无关。相反，pFr-SV(X)感染的培养物持续产生对G418有抗性的定向血红蛋白化红系爆式集落和髓系集落，而pZIP-SV(X)感染的培养物则没有。这些结果表明，pFr-SV(X)和pZIP-SV(X)都能在更原始的多谱系造血祖细胞中稳定整合并表达，并表明当这些细胞分化为定向髓系和红系细胞时，载体的增强子序列会影响转移的新霉素抗性基因的表达。
