Argonaute 2 is a key regulator of maternal mRNA degradation in mouse early embryos.

In mammalian early embryos, the transition from maternal to embryonic control of gene expression requires timely degradation of a subset of maternal mRNAs (MRD). Recently, zygotic genome activation (ZGA)-dependent MRD has been characterized in mouse 2-cell embryo. However, in early embryos, the dynamics of MRD is still poorly understood, and the maternal factor-mediated MRD before and along with ZGA has not been investigated. Argonaute 2 (Ago2) is highly expressed in mouse oocyte and early embryos. In this study, we showed that Ago2-dependent degradation involving RNA interference (RNAi) and RNA activation (RNAa) pathways contributes to the decay of over half of the maternal mRNAs in mouse early embryos. We demonstrated that AGO2 guided by endogenous small interfering RNAs (endosiRNAs), generated from double-stranded RNAs (dsRNAs) formed by maternal mRNAs with their complementary long noncoding RNAs (CMR-lncRNAs), could target maternal mRNAs and cooperate with P-bodies to promote MRD. In addition, we also showed that AGO2 may interact with small activating RNAs (saRNAs) to activate Yap1 and Tead4, triggering ZGA-dependent MRD. Thus, Ago2-dependent degradation is required for timely elimination of subgroups of maternal mRNAs and facilitates the transition between developmental states.

Endo-siRNAs repress expression of SINE1B during in vitro maturation of porcine oocyte.

Approximately 40% of mammalian genome is made of transposable elements (TEs), and during specific biological processes, such as gametogenesis, they may be activated by global demethylation, so strict silencing mechanism is indispensable for genomic stability. Here, we performed small RNA-seq on Dicer1 knockdown (KD) oocytes in pig, and observed short interspersed nuclear elements 1B (SINE1B) derived endogenous small interfering RNAs (endo-siRNAs), termed SINE1B-siRNAs, were significantly decreased and their biogenesis was dependent on Dicer1 and transcript of SINE1B. Furthermore, by injection of mimics and inhibitors of the SINE1B-siRNAs into germinal vesicle-stage (GV-stage) oocytes, we found the maturation rate was significantly decreased by SINE1B-siRNAs, indicating the SINE1B-siRNAs are indispensible for in vitro maturation (IVM) of porcine oocyte. To figure out the mechanism, we checked the expression pattern and DNA methylation status of SINE1B during IVM of porcine oocytes, and demonstrated the SINE1B-siRNAs could repress SINE1B expression induced by hypomethylation at a post-transcriptional level. Our results suggest that during gametogenesis when the erasure of DNA methylation occurs, endo-siRNAs act as a chronic response to limit retrotransposon activation.

Production of transgenic broilers by non-viral vectors via optimizing egg windowing and screening transgenic roosters.

The generation of transgenic chickens is of both biomedical and agricultural significance, and recently chicken transgenesis technology has been greatly advanced. However, major issues still exist in the efficient production of transgenic chickens. This study was designed to optimize the production of enhanced green fluorescence protein (EGFP)-transgenic broilers, including egg windowing at the blunt end (air cell) of egg, and the direct transfection of circulating primordial germ cells by microinjection of the Tol2 plasmid-liposome complex into the early embryonic dorsal aorta. For egg windowing, we discovered that proper manipulation of the inner shell membrane at the blunt end could improve the rate of producing G0 transgenic roosters. From 27 G0 roosters, we successfully collected semen with EGFP-positive sperms from 16 and 19 roosters after direct fluorescence observation and fluorescence-activated cell sorting analyses (13 detected by both methods), respectively. After artificial insemination using the G0 rooster with the highest number of EGFP fluorescent sperm, one G1 EGFP transgenic broiler (1/81, 1.23%) was generated. Our results indicate that appropriate egg windowing and screening of potentially transgene-positive roosters can improve the production of germline-transmitted transgenic birds.

[Latest progress on the chicken genome project].

The publication of draft sequence of the chicken genome in early 2004 marks expressly the start of functional genomics of poultry. Chicken is not only a widely raised economic farm animal, but also a valuable model organism for the study of life sciences. The draft sequence of the chicken genome has significant impact on both animal breeding and basic biological research. The current progress of the chicken genome research is reviewed in this paper, which includes data from the chicken genome, its physical map, genetic linkage map and comparative genome map, as well as expressed sequence tags and bioinformatics. Potential applications of chicken genome research are also envisaged.