Highly efficient gene transfer into adult ventricular myocytes by recombinant adenovirus.
Highly efficient gene transfer into adult ventricular myocytes by recombinant adenovirus.
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DOI：
10.1172/jci116577
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发表时间：
1993-07
期刊：
影响因子：
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通讯作者：
L. Kirshenbaum;W. Maclellan;W. Mazur;B. A. French;M. Schneider
中科院分区：
文献类型：
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作者：
L. Kirshenbaum;W. Maclellan;W. Mazur;B. A. French;M. Schneider
出于令人信服的技术原因，迄今为止，对控制分化心脏表型的机制的分子解剖主要是在新生儿心室肌细胞中进行的。为了规避其他方法的预期局限性，本研究的目的是确定复制缺陷型腺病毒是否能够将基因有效转移到培养的成体心肌细胞中。铺板后 24 小时，用含有巨细胞病毒立即早期启动子驱动的 lacZ 报告基因的 5 型腺病毒感染成年大鼠心室肌细胞，并在感染后 48 小时测定 β-半乳糖苷酶的存在。 lacZ+杆状肌细胞的频率在4 x 10(5)斑块形成单位(PFU)时是最大的一半，并且在1 x 10(8)PFU时接近90%。未感染的细胞和感染lacZ病毒的细胞保持无色。 β-半乳糖苷酶活性与 lacZ+ 细胞的比例一致，并且取决于外源 lacZ 基因。在 10(8) PFU/培养皿时，细胞数量、形态和活力均与未感染的细胞相当。因此，成年心室肌细胞适合用重组腺病毒进行有效的基因转移。腺病毒基因转移的相对均匀性应有助于测试以确定假定的调节剂对病毒修饰的成体心室肌细胞的内源基因和基因产物的影响。
Molecular dissection of mechanisms that govern the differentiated cardiac phenotype has, for cogent technical reasons, largely been undertaken to date in neonatal ventricular myocytes. To circumvent expected limitations of other methods, the present study was initiated to determine whether replication-deficient adenovirus would enable efficient gene transfer to adult cardiac cells in culture. Adult rat ventricular myocytes were infected, 24 h after plating, with adenovirus type 5 containing a cytomegalovirus immediate-early promoter-driven lacZ reporter gene and were assayed for the presence of beta-galactosidase 48 h after infection. The frequency of lacZ+ rod-shaped myocytes was half-maximal at 4 x 10(5) plaque-forming units (PFU) and approached 90% at 1 x 10(8) PFU. Uninfected cells and cells infected with lacZ- virus remained colorless. Beta-galactosidase activity concurred with the proportion of lacZ+ cells and was contingent on the exogenous lacZ gene. At 10(8) PFU/dish, cell number, morphology, and viability each were comparable to uninfected cells. Thus, adult ventricular myocytes are amenable to efficient gene transfer with recombinant adenovirus. The relative uniformity for gene transfer by adenovirus should facilitate tests to determine the impact of putative regulators upon the endogenous genes and gene products of virally modified adult ventricular muscle cells.