Expression levels of the PiT-2 receptor explain, in part, the gestational age-dependent alterations in transduction efficiency after in utero retroviral-mediated gene transfer.
Expression levels of the PiT-2 receptor explain, in part, the gestational age-dependent alterations in transduction efficiency after in utero retroviral-mediated gene transfer.
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DOI：
10.1002/jgm.2607
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发表时间：
2012-03
影响因子：
3.5
通讯作者：
Porada, Christopher D.
中科院分区：
文献类型：
--
作者：
Ozturk, Ferhat;Park, Paul J.;Tellez, Joseph;Colletti, Evan;Eiden, Maribeth V.;Almeida-Porada, Graca;Porada, Christopher D.
使用逆转录病毒介导的基因转移（GT）来治疗人类疾病的根本障碍是在临床相关的人类细胞中已经实现的相对低的转导水平。我们先前表明，在子宫内进行GT克服了这一障碍，并导致多个胎儿器官内的显著水平的转导，不同的组织在不同的发育阶段表现出最佳的转导。我们进行了本研究，旨在阐明这种年龄依赖性转导的机制，测试我们假设可能负责的两个因素：（i）GT时组织的增殖状态和（ii）嗜酸性PiT-2受体的表达水平。对来自不同发育阶段的绵羊的组织进行免疫荧光，以评估作为年龄的函数的每个器官内的主要细胞的增殖状态。在开发了酶联免疫吸附试验（ELISA）和定量逆转录链反应（qRT-PCR）测定后，我们分别在蛋白质和mRNA水平上定量PiT-2的表达。所获得的结果表明，在胎儿GT的时间器官的增殖状态是不是主要的决定因素支配转导效率。相反，我们的ELISA和qRT-PCR分析表明，PiT-2 mRNA和蛋白水平随胎龄变化，与观察到的转导效率差异相关。本研究的结果解释了年龄相关的差异，我们以前观察到的转导效率后，在子宫内GT。他们还表明，它可能是可能的，以实现相对选择性GT特定组织进行子宫内GT时，PiT-2的水平是最大的，在所需的靶器官。
A fundamental obstacle to using retroviral-mediated gene transfer (GT) to treat human diseases is the relatively low transduction levels that have been achieved in clinically relevant human cells. We previously showed that performing GT in utero overcomes this obstacle and results in significant levels of transduction within multiple fetal organs, with different tissues exhibiting optimal transduction at different developmental stages. We undertook the present study aiming to elucidate the mechanism for this age-dependent transduction, testing the two factors that we hypothesized could be responsible: (i) the proliferative status of the tissue at the time of GT and (ii) the expression level of the amphotropic PiT-2 receptor. Immunofluorescence was performed on tissues from sheep of varying developmental stages to assess the proliferative status of the predominant cells within each organ as a function of age. After developing an enzyme-linked immunosorbent assay (ELISA) and a quantitative reverse transcription chain reaction (qRT-PCR) assay, we then quantified PiT-2 expression at the protein and mRNA levels, respectively. The results obtained indicate that the proliferative status of organs at the time of fetal GT is not the major determinant governing transduction efficiency. By contrast, our ELISA and qRT-PCR analyses demonstrated that PiT-2 mRNA and protein levels vary with gestational age, correlating with the observed differences in transduction efficiency. The findings of the present study explain the age-related differences that we previously observed in transduction efficiency after in utero GT. They also suggest it may be possible to achieve relatively selective GT to specific tissues by performing in utero GT when levels of PiT-2 are maximal in the desired target organ.
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影响因子：
5.4
作者：
WILSON, CA;EIDEN, MV
通讯作者：
EIDEN, MV
影响因子：
3.4
作者：
Boyer, CJC;Baines, AD;Béliveau, R
通讯作者：
Béliveau, R
影响因子：
5.4
作者：
Feldman, SA;Farrell, KB;Eiden, MV
通讯作者：
Eiden, MV
影响因子：
5.4
作者：
Chien, ML;Foster, JL;Garcia, JV
通讯作者：
Garcia, JV
影响因子：
5.4
作者：
MILLER, DG;MILLER, AD
通讯作者：
MILLER, AD