生物
遗传增强
表皮生长因子
离体
病毒载体
信号转导
分子生物学
转基因
移植
生长因子
癌症研究
肝细胞生长因子
细胞培养
细胞生物学
转导(生物物理学)
肝细胞
基因
体内
免疫学
体外
受体
内科学
遗传学
医学
生物化学
重组DNA
作者
Michael Rothe,Ina Rittelmeyer,Marcus Iken,Urda Rüdrich,Axel Schambach,Silke Glage,Michael P. Manns,Christopher Baum,Michael Bock,Michael Ott,Ute Modlich
出处
期刊:Gene Therapy
[Springer Nature]
日期:2011-08-18
卷期号:19 (4): 425-434
被引量:23
摘要
Partial resistance of primary mouse hepatocytes to lentiviral (LV) vector transduction poses a challenge for ex vivo gene therapy protocols in models of monogenetic liver disease. We thus sought to optimize ex vivo LV gene transfer while preserving the hepatocyte integrity for subsequent transplantation into recipient animals. We found that culture media supplemented with epidermal growth factor (EGF) and, to a lesser extent, hepatocyte growth factor (HGF) markedly improved transduction efficacy at various multiplicities of infection. Up to 87% of primary hepatocytes were transduced in the presence of 10 ng EGF, compared with ~30% in standard culture medium (SCMs). The increased number of transgene-expressing cells correlated with increased nuclear import and more integrated pro-viral copies per cell. Higher LV transduction efficacy was not associated with proliferation, as transduction capacity of gammaretroviral vectors remained low (<1%). Finally, we developed an LV transduction protocol for short-term (maximum 24 h) adherent hepatocyte cultures. LV-transduced hepatocytes showed liver repopulation capacities similar to freshly isolated hepatocytes in alb-uPA mouse recipients. Our findings highlight the importance of EGF for efficient LV transduction of primary hepatocytes in culture and should facilitate studies of LV gene transfer in mouse models of monogenetic liver disease.
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