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Potential application of dominant negative retinoicacid receptor genes for ex vivo expansion ofhematopoietic stem cells

机译：显性负视黄酸受体基因在造血干细胞离体扩增中的潜在应用

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It is difficult to expand hematopoietic stem cells ex vivo by stimulation with hematopoietic cytokines, since any cytokine thus far tested induces differentiation as well as growth. It is therefore important to consider how to inhibit differentiation of hematopoietic stem cells during ex vivo culture. For this purpose, we have constructed retroviral vectors expressing dominant-negative retinoic acid receptor- (RAR ) genes. The immature hematopoietic cell lines 32D and FDCPmix, transduced with the dominant-negative RAR -expressing vectors, remained blastic or promyelocytic and continued to grow without differentiation even under the differentiation-inducing conditions. This block of differentiation could be overcome by treatment with all-trans retinoic acid, suggesting that the transduced cells still retained their differentiating ability. This leads to the possible application of dominant-negative RAR ? genes for the ex vivo expansion of hematopoietic stem cells in combination with hematopoietic cytokines. For clinical applications, however, dominant-negative RAR ? genes should be removed from host cells after ex vivo expansion. We have, therefore, incorporated two loxP sites on either side of the RAR gene in each vector so that removal of the integrated differentiation-blocking RAR ? genes from the transduced cells could be achieved using the Cre recombinase/loxP system (reversible integration of a gene of interest). We are investigating efficient ways to introduce the Cre recombinase into host cells.
机译：通过用造血细胞因子刺激离体扩增造血干细胞是困难的，因为迄今为止测试的任何细胞因子都诱导分化以及生长。因此，重要的是考虑在体外培养期间如何抑制造血干细胞的分化。为此，我们构建了表达显性负视黄酸受体（RAR）基因的逆转录病毒载体。用表达显性阴性RAR的载体转导的未成熟的造血细胞系32D和FDCPmix，即使在分化诱导条件下，仍保持着增生或早幼粒细胞的状态，并持续生长而没有分化。通过用全反式视黄酸处理可以克服这种分化障碍，这表明转导的细胞仍然保留了它们的分化能力。这导致可能使用显性负RAR。造血干细胞与造血细胞因子联合体外扩增的基因。对于临床应用，显性阴性RAR？离体扩增后，应从宿主细胞中去除基因。因此，我们在每个载体中在RAR基因的任一侧掺入了两个loxP位点，以便除去整合的分化阻断RARα。可以使用Cre重组酶/ loxP系统（目的基因的可逆整合）实现来自转导细胞的基因。我们正在研究将Cre重组酶引入宿主细胞的有效方法。

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