190 SUPPRESSION OF A TRANSCRIPTION FACTOR MSX1 GENE IN BOVINE PREIMPLANTATION EMBRYOS AND ITS EFFECT ON mRNA, PROTEIN, AND EXPRESSION OF DOWNSTREAM GENES

Abstract

MSX1 is a transcription factor gene that orchestrates gene expression and regulates cell growth, proliferation, differentiation, cell-to-cell communication, and the apoptotic pathway during pattern formation in vertebrate embryogenesis. However, its role in bovine preimplantation embryo is not known. Here we aim to investigate the effects of suppressing MSX1 transcript on the development of in vitro-produced bovine embryos, study the expression of mRNA and protein products of the gene, and identify downstream genes using microarray analysis. In the first experiment, IVP zygotes were injected with 341 bp-long dsRNA (LdsRNA) (n = 384), 19 bp small interfering RNA (siRNA) (n = 374), and scrambled sequence RNA (scRNA) (n = 388). Uninjected zygotes (n = 313) were used as control. Developmental phenotype data were collected during culture until Day 8. The mRNA and protein expression levels of the different treatment groups were validated at the 8-cell and blastocyst stages using quantitative real-time PCR and immunohistochemistry, respectively. Developmental phenotype and mRNA data were analyzed using ANOVA under statistical package SPSS (SPSS, Inc., Chicago, IL, USA). In the second experiment, custom SMARTpool siRNA (Dharmacon Inc., Chicago, IL, USA) targeting bovine MSX1 (NM_174798) was used for microinjection together with siRNA and uninjected control. Following treatment at zygote stage, 8-cell embryos were used for mRNA isolation and subsequent array hybridization using bovine cDNA array containing 2000 clones. Array data analysis was performed using statistical analysis of microarray (SAM) procedure. While 33% and 29% of the zygotes from the control and scRNA treatment groups, respectively, reached blastocyst stage, only 20% and 19% of the zygotes from the LdsRNA and siRNA treatment groups, respectively, reached the same stage. Injection of LdsRNA and siRNA at the zygote stage reduced the mRNA expression level by 52% and 33% at the 8-cell stage and by 77% and 87%, respectively, at the blastocyst stage as compared to the control. Similarly, cellular protein expression levels in LdsRNA- and siRNA-injected treatment groups were found to be lower than the control groups at each stage. In all cases, injection of scRNA had no effect on mRNA and protein levels. SAM analysis revealed that, of the total 2000 clones, 3.5% and 5.4% were found to be differentially expressed in embryos injected with SMARTpool and siRNA, respectively, compared to the control. Genes involved in various activities including transcription factors (ALF), cell growth (BMP-15), metabolism (RIOK3), and cytokinasis (AURKA) were found to be down-regulated in 8-cell embryos treated with SMARTpool siRNA compared to the controls. On the other hand, genes involved in protein synthesis (RPL23), energy metabolism (COQ1), cell growth (MNS1) and skeletal development (LGALS3) were found to be upregulated in the same samples. In conclusion, suppression of MSX1 at the mRNA and protein level significantly affected the development of bovine embryos, and our study revealed list of downstream genes regulated by the activity of MSX1.