The effects of miRNA-214 knockout on the embryonic and larval development of col10a1 : nlGFP and osx: mcherry double transgenic medaka (Oryzias latipes)

Abstract

MicroRNA (miRNAs) are highly conserved, non-coding RNAs that regulate gene expression at post-transcriptional level. MiRNA-214 (miR-214) is highly conserved among vertebrate, it has been shown to play multiple roles that implicated in diverse physiological functions, including development of bone, muscle, heart and various human cancers. This project aims to study the role of miR-214 in vivo using a miR-214 knockout (KO) col10a1:nlGFP/ osx:mcherry double transgenic Japanese medaka (Oryzias latipes). It is hypothesized that knockout of miR-214 in medaka would alleviate the suppressive effect on target genes expression, leading to abnormal development and bone formation in early embryo and larvae. F3 generation of miR-214 KO medaka lines were obtained by crossing the F2 heterozygous miR-214 KO medaka, resulting in offspring with three different genotypes. The genotypes of medaka was determined by miR-214 mutant screening and genetic sequencing. The approach of phenotype assessment was divided into two parts, the primary level of phenotype assessment aimed to find out abnormalities of miR-214 KO medaka, while the secondary level of phenotype assessment evaluated and quantified abnormalities in four categories, including disrupted development, circulatory system development, locomotor activity and bone development. Only mild abnormalities were found in heterozygous mutant, while homozygous mutant showed a wide ranges of abnormal phenotype ranging from mild to lethal. Abnormal phenotypes were observed in different organ systems, such as defect in cardiovascular system, bone deformity, and abnormal pigmentation development in eye and gallbladder, which suggested that miR-214 might play widespread roles in organism through simultaneously targeting on multiple gene to regulate gene expression.