BACKGROUND Viral vectors are invaluable tools to transfer genes and/or regulatory sequences into differentiated cells such as pancreatic cells. To date, several kinds of viral vectors have been used to transduce different pancreatic cell types, including insulin-producing β cells. However, few studies have used vectors derived from « simple » retroviruses, such as avian α- or mouse γ-retroviruses, despite their high experimental convenience. Moreover, such vectors were never designed to specifically target transgene expression into β cells. RESULTS We here describe two novel α- or SIN (Self-Inactivating) γ-retrovectors containing the RIP (Rat Insulin Promoter) as internal promoter. These two retrovectors are easily produced in standard BSL2 conditions, rapidly concentrated if needed, and harbor a large multiple cloning site. For the SIN γ-retrovector, either the VSV-G (pantropic) or the retroviral ecotropic (rodent specific) envelope was used. For the α-retrovector, we used the A type envelope, as its receptor, termed TVA, is only naturally present in avian cells and can efficiently be provided to mammalian β cells through either exogenous expression upon cDNA transfer or gesicle-mediated delivery of the protein. As expected, the transgenes cloned into the two RIP-containing retrovectors displayed a strong preferential expression in β over non-β cells compared to transgenes cloned in their non-RIP (CMV- or LTR-) regulated counterparts. We further show that RIP activity of both retrovectors mirrored fluctuations affecting endogenous INSULIN gene expression in human β cells. Finally, both α- and SIN γ-retrovectors were extremely poorly mobilized by the BXV1 xenotropic retrovirus, a common invader of human cells grown in immunodeficient mice, and, most notably, of human β cell lines. CONCLUSION Our novel α- and SIN γ-retrovectors are safe and convenient tools to stably and specifically express transgene(s) in mammalian β cells. Moreover, they both reproduce some regulatory patterns affecting INSULIN gene expression. Thus, they provide a helpful tool to both study the genetic control of β cell function and monitor changes in their differentiation status.

中文翻译：

---

用于在胰腺β细胞系中安全转导和特异性转基因表达的新型α和SINγ逆转录载体。

背景技术病毒载体是将基因和/或调节序列转移到分化细胞如胰腺细胞中的宝贵工具。迄今为止，几种病毒载体已被用于转导不同的胰腺细胞类型，包括产生胰岛素的β细胞。但是，尽管它们具有很高的实验便利性，但很少有研究使用源自“简单”逆转录病毒的载体，例如禽α-或小鼠γ-逆转录病毒。而且，从未设计过将此类载体特异性地靶向转基因表达进入β细胞。结果我们在此描述了两种新型的R或SIN（自灭活）γ逆转录载体，其中含有RIP（大鼠胰岛素促进剂）作为内部启动子。这两个逆转录载体可在标准BSL2条件下轻松生产，并在需要时迅速浓缩，并具有大量的多克隆位点。对于SINγ逆向载体，可以使用VSV-G（泛嗜性）或逆转录病毒生态亲和性（啮齿动物特异性）包膜。对于α逆转录载体，我们使用了A型包膜，因为其受体（称为TVA）仅天然存在于禽类细胞中，并且可以通过cDNA转移时的外源表达或囊泡介导的Aβ转运有效地提供给哺乳动物β细胞。蛋白质。如预期的那样，与在非RIP（CMV-或LTR-）调控的对应物中克隆的转基因相比，克隆到两个包含RIP的逆转录载体中的转基因在β中显示出比非β细胞更强的优先表达。我们进一步表明，两个逆转录载体的RIP活性反映了影响人β细胞内源性INSULIN基因表达的波动。最后，BXV1异种性逆转录病毒（免疫缺陷小鼠中生长的人类细胞，尤其是人类β细胞系）的常见入侵者，α和SINγ逆转录载体的动员能力都非常差。结论我们新颖的α和SINγ逆转录载体是在哺乳动物β细胞中稳定和特异性表达转基因的安全便捷工具。而且，它们都重现了一些影响INSULIN基因表达的调控模式。因此，它们为研究β细胞功能的遗传控制和监测其分化状态的变化提供了有用的工具。它们都重现了一些影响INSULIN基因表达的调控模式。因此，它们为研究β细胞功能的遗传控制和监测其分化状态的变化提供了有用的工具。它们都重现了一些影响INSULIN基因表达的调控模式。因此，它们为研究β细胞功能的遗传控制和监测其分化状态的变化提供了有用的工具。