Fabp4-Cre-mediated deletion of the miRNA-processing enzyme Dicer causes mouse embryonic lethality

Abstract

MicroRNAs (miRNAs) are increasingly being recognized as important regulators of gene expression through the inhibition of effective mRNA translation via imperfect base pairing with the 30-untranslated region of target mRNAs in animals. Emerging evidence suggests that miRNA-mediated gene regulation represents a fundamental layer of genetic programming at the post-transcriptional level and has diverse functional roles, including development, differentiation and homeostasis [1, 2]. Recently accumulated studies indicate that miRNAs are also involved in adipocyte development in vitro [3–13] and that the expression of serum miRNAs is changed during type 2 diabetes development [14]. Dicer is required for the processing of mature and functional miRNAs. Therefore, deletion of Dicer provides a genetic test for the relevance of miRNAs in mammalian development. However, conventional Dicer knockout causes mouse embryonic lethality. To test whether miRNAs are involved in the adipocyte development and function in vivo, we recently generated adipocyte-specific miRNA knockout mice by crossing mice with loxp-flanked locus of Dicer generated by Dr. Harfe’s group [15] with Fabp4-Cre transgenic mice [B6.Cg-Tg(Fabp4-Cre), #005069, purchased from the Jackson Laboratory] containing the Cre recombinase gene under transcriptional control of Fabp4 (Fatty Acid Binding Protein 4, also called aP2) promoter. After the first crossing, DicerFabp4-Cre mice were bred with Dicer Fabp4-Cre mice. Based on Mendel’s Laws (law of segregation), we expected four different genotype mice: homozygous Dicer knockout (KO) mice (DicerFabp4Cre), wild-type mice (WT) (DicerFabp4-Cre and DicerFabp4-Cre), and heterozygous mice (Dicer Fabp4-Cre), and 50% mice could be Cre mice. A total of 93 live-born offspring were obtained from 13 litters, but only 35.5% were Cre mice. As shown in Table 1, among 93 offspring, there were 60 (64.5%) WT and 33 (35.5%) heterozygous mice, but no homozygous KO mice were identified. Thus, these findings highly suggested that DicerFabp4-Cre KO mice may die before the birth. To further support this notion, the embryos were collected at day E14. A total of 28 embryos were analyzed from 5 litters. All embryos had the regular size, and the gross appearance was normal. As shown in Table 1, these embryos had four different types of genotype, which was consistent with Mendel’s Laws. The relative proportion of homozygous KO embryos was *21%. The percentages of Cre and Cre mice were 57 and 43%, respectively. Thus, Fabp4-Cre-mediated Dicer deletion causes mouse embryonic lethality in the late-stage gestation. Mudhasani et al. [16] recently also generated adipocytespecific miRNA knockout mice. They crossed mice with Dicer conditional alleles [17] with Fabp4-Cre transgenic Dong-Mei Meng, Luan Wang and Jian-Rui Xu contributed equally to this study.