Nonspecific suppression of [3H]thymidine incorporation by "control" oligonucleotides.-猫眼课题宝

“对照”寡核苷酸对[3H]胸苷掺入的非特异性抑制。

DOI：

发表时间：

1992

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Antisense Research and Development

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中科院分区：

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作者：

S. Matson;A. Krieg

磷酸二酯寡核苷酸在脾细胞培养物中快速降解。进行本研究以确定从此类寡核苷酸降解中释放的胸苷是否可以被重新利用并与[3H]胸苷掺入竞争，从而引起“增殖”测定的非特异性抑制。我们对有丝分裂原刺激的小鼠脾细胞的研究表明，含有胸苷的“对照”寡核苷酸可以对[3H]胸苷掺入产生90%以上的抑制。这种抑制作用通常取决于寡核苷酸内胸苷的位置：具有 3' 末端胸苷的寡核苷酸比胸苷位于 5' 末端的寡核苷酸引起更多的抑制。所有寡核苷酸都会对[3H]尿苷掺入产生适度但可变的抑制。此外，[3H]胸苷掺入甚至被不含胸苷的寡核苷酸部分抑制。我们建议使用[3H]胸苷掺入测定来评估反义效应的研究人员谨慎行事。谨慎的做法是使用具有相同数量和位置的胸苷碱基的对照寡核苷酸，并通过其他细胞增殖测量来确认[3H]胸苷掺入测定。

Phosphodiester oligonucleotides are rapidly degraded in spleen cell cultures. The present studies were conducted to determine whether thymidine released from degradation of such oligonucleotides could be reutilized and compete with [3H]thymidine incorporation, thereby causing nonspecific inhibition of "proliferation" assays. Our studies in mitogen-stimulated mouse spleen cells demonstrate that "control" oligonucleotides that contain thymidine can cause more than 90% inhibition of [3H]thymidine incorporation. This inhibitory effect was generally dependent on the location of the thymidine within the oligonucleotide: oligonucleotides that had 3'-terminal thymidine(s) caused more suppression than those in which thymidines were at the 5' end. All oligonucleotides caused a modest but variable inhibition of [3H]uridine incorporation. Furthermore, [3H]thymidine incorporation was partially inhibited even by oligonucleotides that did not contain thymidine. We propose that investigators who use [3H]thymidine incorporation assays to assess antisense effects do so with caution. It may be prudent to use control oligonucleotides with the same number and location of thymidine bases and to confirm [3H]thymidine incorporation assays with other measures of cell proliferation.

Antiviral effect of an oligo(nucleoside methylphosphonate) complementary to the splice junction of herpes simplex virus type 1 immediate early pre-mRNAs 4 and 5.