New manuscript accepted
Computational identification of microRNA gene loci and precursor microRNA sequences in CHO cell lines
Hackl M, Jadhav V, Jakobi T, Rupp O, Brinkrolf K, Goesmann A, Pühler A, Noll T, Borth N, Grillari J
Journal of Biotechnology, accepted
MicroRNAs (miRNAs) have recently entered Chinese hamster ovary (CHO) cell culture technology, due to their severe impact on the regulation of cellular phenotypes. Applications of miRNAs that are envisioned range from biomarkers of favorable phenotypes to cell engineering targets. Especially the latter application, however, requires a profound knowledge of miRNA sequences and their genomic organization, which exceeds the currently available information of 398 conserved mature CHO miRNA sequences. Based on these recently published sequences and two independent CHO-K1 genome assemblies, this publication describes the computational identification of CHO miRNA genomic loci. Using BLAST alignment, a total of 354 miRNAs were confidently mapped to the reference genome, and subsequently assigned to 230 distinct genomic miRNA loci, as approximately 50% of miRNAs represented matching pairs of 5' and 3' miRNAs. Sequences of these 230 precursor-miRNAs were extracted from both reference genomes and cross-compared. The genomic loci and pre-miRNA sequences for 354 expressed mature miRNAs were submitted to the miRBase miRNA repository. As a proof-of-principle for the usability of the published genomic loci, four polycistronic miRNA cluster were chosen for PCR amplification using CHO-K1 and DHFR (-) genomic DNA. Overall, the here presented information on the genomic context in which miRNAs are expressed in CHO will simplify the development of tools employing stable overexpression or deletion of miRNAs, allow the identification of miRNA promoters and improve detection methods such as microarrays.