Johnson L G, Pickles R J, Boyles S E, Morris J C, Ye H, Zhou Z, Olsen J C, Boucher R C
CF/Pulmonary Research and Treatment Center, University of North Carolina at Chapel Hill 27599, USA.
Hum Gene Ther. 1996 Jan;7(1):51-9. doi: 10.1089/hum.1996.7.1-51.
Primary cultures of airway epithelia were used to evaluate variables pertinent to adenovirus (Ad)-mediated gene transfer efficiency and efficacy including: (i) Ad-vectors with different promoters, (ii) the duration of vector incubation with cells, (iii) the concentration and depth of vector-containing medium at constant multiplicity of infection (moi) (10(3)), and (iv) the relative sensitivity of reverse transcription polymerase chain reaction (RT-PCR) versus functional analysis for the detection of transduced cystic fibrosis transmembrane conductance regulator (CFTR). An Ad5-lacZ vector with a cytomegalovirus (CMV) enhancer/promoter transduced the greatest amount of beta-galactosidase (beta-Gal) activity, while an Ad2-lacZ vector with an E1a enhancer/promoter transduced the least. Ad5-lacZ vectors with the Rous sarcoma virus (RSV), E1a/RSV, or CMV enhancer/beta-actin (CB) promoters transduced intermediate levels of beta-Gal. Optimal gene transfer efficiency was detected with a 4-8 hr incubation of Ad5-CMVlacZ with cells, although optimal CFTR Cl-transport function was detectable after only a 30 min incubation of Ad5-CBCFTR with cells, consistent with correction of > or = 6-10% of cells in the epithelial sheet. Ad5-CBCFTR transduction of CF airway epithelial cells (moi = 10(3)) was optimal when higher concentrations, lower volumes, or smaller depths of vector-containing medium were utilized. RT-PCR was at least 100-fold more sensitive for the detection of transduced CFTR than functional analysis, and could detect as few as 0.001% Ad5-CBCFTR-infected CF cells admixed with uninfected CF cells. In summary, the variables studied clearly affect the efficiency of Ad-mediated gene transfer in vitro and potentially in vivo. They also suggest that RT-PCR is a poor marker of gene transfer efficiency and efficacy.
气道上皮细胞的原代培养用于评估与腺病毒（Ad）介导的基因转移效率和效力相关的变量，包括：（i）具有不同启动子的Ad载体；（ii）载体与细胞孵育的持续时间；（iii）在恒定感染复数（moi）（10³）下含载体培养基的浓度和深度；以及（iv）逆转录聚合酶链反应（RT-PCR）与功能分析在检测转导的囊性纤维化跨膜电导调节因子（CFTR）方面的相对敏感性。带有巨细胞病毒（CMV）增强子/启动子的Ad5-lacZ载体转导的β-半乳糖苷酶（β-Gal）活性最高，而带有E1a增强子/启动子的Ad2-lacZ载体转导的最少。带有劳斯肉瘤病毒（RSV）、E1a/RSV或CMV增强子/β-肌动蛋白（CB）启动子的Ad5-lacZ载体转导的β-Gal水平中等。Ad5-CMVlacZ与细胞孵育4-8小时可检测到最佳基因转移效率，尽管Ad5-CBCFTR与细胞仅孵育30分钟后即可检测到最佳CFTR Cl⁻转运功能，这与上皮片中≥6-10%的细胞得到校正一致。当使用更高浓度、更低体积或更小深度的含载体培养基时，Ad5-CBCFTR对CF气道上皮细胞（moi = 10³）的转导效果最佳。RT-PCR检测转导的CFTR的敏感性比功能分析至少高100倍，并且能够检测到与未感染的CF细胞混合的低至0.001%的Ad5-CBCFTR感染的CF细胞。总之，所研究的变量明显影响体外以及可能在体内的Ad介导的基因转移效率。它们还表明RT-PCR不是基因转移效率和效力的良好标志物。
