It is estimated that more than 30% of all protein-coding genes in the human genome are regulated by miRNAs. This significant role has resulted in a considerable amount of attention being paid to this family of noncoding RNA molecules.
Select Biosciences’ recent “RNAi and MiRNA Europe” meeting featured discussions on cutting-edge technologies for integrating miRNAs into the genome and for functionally analyzing expression. Insights to help unravel the complex picture of miRNA interactions, including modeling miRNA networks using high-tech tools of space-age cryptography and communications signal analysis, were also presented.
DNA integration is a powerful technology that enables disruption, modification, or replacement of an endogenous locus with a new gene. That same technology can also be utilized with miRNA genes.
In the past, most genes were randomly integrated into the host’s genome. “The problem is that this can produce off-target effects,” noted Kevin Forbes, Ph.D., senior R&D scientist at Sigma-Aldrich. Dr. Forbes said that the company’s CompoZr™ Zinc Finger Nuclease (ZFN) technology allows the site-specific integration of any gene, including those of miRNA.
“We’ve seen a lot of interest in this new technology because most people have used lentiviral systems that integrate randomly. ZFN technology employs a pair of well-validated nuclear ‘safe harbor’ sites. We know exactly where in the genome it goes and can rule out positional effects.”
Dr. Forbes has expressed miRNA genes from the human genome utilizing the company’s CompoZr Targeted Integration Kit that places a specified gene into the adeno-associated virus (AAVS1) site on human chromosome 19. ZFNs create a double-stranded break that can be repaired by the supplied pZDonor plasmid, since it has homologous arms to the AAVS1 site at either end of a multiple cloning site. Thus, the repair takes place and inserts the gene between the homologous arms.
Scientists at Sigma-Aldrich have focused many of their efforts on miRNAs related to cancer metastasis. “Using this technology we provided the first proof-of-concept that we can express specific miRNAs at a known human locus. We expect many applications for both research and therapeutics. Certainly, a key benefit is that we can now provide a well-characterized site found in every human cell for targeted integration.”