49 REPROGRAMMING OF MICRO RNA IN BOVINE CLONED ELONGATED EMBRYOS

Abstract

MicroRNAs (miRNA) are key regulators of gene expression in vertebrate development; however, little information exists about their expression in pre-implantation mammalian embryos. We studied the expression of miRNA in bovine cloned and IVF-elongated embryos using microarrays and quantitative RT-PCR (qRT-PCR). Day-7 IVF or handmade cloned blastocysts from an adult cell line that previously yielded live cloned calves were transferred to synchronized heifers and recovered at Day 17 as elongated embryos. MicroRNAs were purified using miRVANA kit+Flash Page fractionator column (Ambion, Austin, TX, USA) coupled to amine reactive dyes CyDye™ or used in qRT-PCR. Arrays were custom-made from 271 mice, human, and rat and 104 predicted miRNAs. After overnight hybridization, slides were scanned and data analyzed. Only miRNAs showing hybridization in 3 out of 6 features per slide were considered to be expressed. MicroRNAs were simultaneously converted to complementary (c)DNA and elongated using miRNA-specific DNA-oligo with 5′ overhang and primer extension by reverse transcription, followed by SYBR Green qPCR with another miRNA-specific primer and an excess of universal forward and reverse primers. The qPCR were normalized against 5S rRNA. Data were analyzed using Kruskal-Wallis nonparametric test for P-values < 0.05. Cloned (n = 48) and IVF (n = 28) blastocysts were transferred into 10 and 4 surrogate cattle, and 42 elongated embryos were recovered; 13 of these with embryonic disc were used for miRNA extraction and analysis. In experiment 1, miRNA profiling (1 slide) from 4 cloned embryos and donor cells yielded 34 and 22 miRNAs expressed in the embryos and cells, respectively. In experiment 2 (differential expression; 3 replicates), 39 miRNAs were expressed in cloned embryos and 32 in IVF embryos. Fifteen of these miRNAs were differentially expressed: 7 were up-regulated in the cloned elongated embryos and 8 in the elongated IVF embryos. The profiles of miRNA expression of cloned embryos, IVF embryos, and the donor cells were compared and reprogramming patterns analyzed. Reprogramming means similar expression among cloned and IVF embryos, but different from the somatic cells. Overall, 50 miRNAs were subjected to nuclear reprogramming; 31 (62%) were reprogrammed correctly, 10 aberrantly (20%), and 9 were not reprogrammed (18%). Most of the aberrantly reprogrammed miRNAs (90%) were switched on after nucleus transfer. Some of the reprogrammed miRNAs clustered in the same genomic location. Based on microarray data, 3 miRNAs over-expressed in the IVF embryos (let7-b, miR-200c, and miR-24), and 1 equally expressed (miR-16) and 2 over-expressed in the cloned embryos (miR-21 and miR-103) were analyzed by qRT-PCR. Expression of all the assayed miRNAs except miR-200c correlated adequately with microarray data (Pearson coefficient of correlation R² = 0.9; P = 0.01). Here we present the first description of miRNA expression and reprogramming in bovine elongated cloned embryos. This can have profound implications for the understanding of nuclear reprogramming in somatic cloning.