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MicroRNAs (miRNAs) are small, noncoding RNAs of about 22 nucleotides in length and have proven to be useful targets for genetic modifications for desirable phenotypes in the biotech industry. The use of constitutively expressed “miRNA sponge” vectors in which multiple, tandem miRNA-binding sites containing transcripts are transcriptionally regulated by a constitutive promoter for downregulating the levels of endogenous microRNAs in Chinese hamster ovary (CHO) cells has shown to be more advantageous than using synthetic antisense oligonucleotides. The application of miRNA sponges in biotechnological processes, however, could be more effective, if the expression of miRNA sponges could be tuned. In this chapter, we present a method for the generation of stable CHO cell lines expressing a TET-ON-SanDI-miRNA sponge which is in theory expressed only in the presence of an inducer.

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Conditional Knockdown of Endogenous MicroRNAs in CHO Cells Using TET-ON-SanDI Sponge Vectors

  • 实验方案

  • 发表日期：

    2025年

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  • Alan Costello,
  • Nga Lao,
  • Martin Clynes,
  • Niall Barron

摘要

MicroRNAs (miRNAs) are small, noncoding RNAs of about 22 nucleotides in length and have proven to be useful targets for genetic modifications for desirable phenotypes in the biotech industry. The use of constitutively expressed “miRNA sponge” vectors in which multiple, tandem miRNA-binding sites containing transcripts are transcriptionally regulated by a constitutive promoter for downregulating the levels of endogenous microRNAs in Chinese hamster ovary (CHO) cells has shown to be more advantageous than using synthetic antisense oligonucleotides. The application of miRNA sponges in biotechnological processes, however, could be more effective, if the expression of miRNA sponges could be tuned. In this chapter, we present a method for the generation of stable CHO cell lines expressing a TET-ON-SanDI-miRNA sponge which is in theory expressed only in the presence of an inducer.