Rev-dependent lentiviral expression vector
Rev-dependent lentiviral expression vector
复制标题
DOI：
10.1186/1742-4690-4-12
复制
发表时间：
2007-02-07
期刊：
影响因子：
3.3
通讯作者：
Marsh, Jon W.
中科院分区：
文献类型：
--
作者：
Wu, Yuntao;Beddall, Margaret H.;Marsh, Jon W.
背景：HIV 响应表达载体均基于 HIV 启动子，即长末端重复序列 (LTR)。在对早期 HIV 蛋白 Tat 做出反应的同时，LTR 还对细胞激活状态以及发生整合的局部染色质活性做出反应。这可能导致高度的 HIV 独立活性，并限制了基于 LTR 的报告载体在克隆细胞中的使用，在克隆细胞中可以消除异常高表达（HIV 阴性）细胞。特异性的增强将增加表达载体用于检测 HIV 以及 HIV 感染细胞中的实验基因表达的机会。 结果：我们构建了一种表达载体，除了 Tat 响应 LTR 之外，还拥有大量 HIV DNA 序列，其中包括 Rev 响应元件和 HIV 剪接位点，可在人类细胞中有效使用。它还包含一个阅读框，在没有 HIV Rev 的情况下，该阅读框会被细胞剪接活动去除。该载体被整合到慢病毒报告病毒中，从而可以检测活细胞群中复制的 HIV。通过绿色荧光蛋白 (GFP) 报告基因的表达和 HIV 感染后报告基因转录本的 PCR 来测量载体的活性。该载体表现出完全的 HIV 依赖性。结论：与早期开发的 Tat 依赖性表达载体一样，这里描述的 Rev 系统是对 HIV 进化过程的利用。 Rev依赖性的包含使得基于LTR的表达载体高度依赖于复制的HIV的存在。本文报道的这种载体（一种 HIV 依赖性报告病毒）的应用为现有方法（原位 PCR 或 HIV 抗原染色）提供了一种新的替代方法，以识别 HIV 阳性细胞。该载体允许检查活细胞，可以表达用于基础或临床实验的任何基因，并且作为假型慢病毒可以接触大多数细胞类型和组织。
Background: HIV-responsive expression vectors are all based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, the LTR is also responsive to cellular activation states and to the local chromatin activity where the integration has occurred. This can result in high HIV-independent activity, and has restricted the use of LTR-based reporter vectors to cloned cells, where aberrantly high expressing (HIV-negative) cells can be eliminated. Enhancements in specificity would increase opportunities for expression vector use in detection of HIV as well as in experimental gene expression in HIV-infected cells.Results: We have constructed an expression vector that possesses, in addition to the Tat-responsive LTR, numerous HIV DNA sequences that include the Rev-response element and HIV splicing sites that are efficiently used in human cells. It also contains a reading frame that is removed by cellular splicing activity in the absence of HIV Rev. The vector was incorporated into a lentiviral reporter virus, permitting detection of replicating HIV in living cell populations. The activity of the vector was measured by expression of green fluorescence protein (GFP) reporter and by PCR of reporter transcript following HIV infection. The vector displayed full HIV dependency.Conclusion: As with the earlier developed Tat-dependent expression vectors, the Rev system described here is an exploitation of an evolved HIV process. The inclusion of Rev-dependency renders the LTR-based expression vector highly dependent on the presence of replicating HIV. The application of this vector as reported here, an HIV-dependent reporter virus, offers a novel alternative approach to existing methods, in situ PCR or HIV antigen staining, to identify HIV-positive cells. The vector permits examination of living cells, can express any gene for basic or clinical experimentation, and as a pseudo-typed lentivirus has access to most cell types and tissues.