Using a nano-flare probe to detect RNA in live donor cells prior to somatic cell nuclear transfer.

Many transgenes are silenced in mammalian cells (donor cells used for somatic cell nuclear transfer [SCNT]). Silencing correlated with a repressed chromatin structure or suppressed promoter, and it impeded the production of transgenic animals. Gene transcription studies in live cells are challenging because of the drawbacks of reverse-transcription polymerase chain reaction and fluorescence in situ hybridization. Nano-flare probes provide an effective approach to detect RNA in living cells. We used 18S RNA, a housekeeping gene, as a reference gene. This study aimed to establish a platform to detect RNA in single living donor cells using a Nano-flare probe prior to SCNT and to verify the safety and validity of the Nano-flare probe in order to provide a technical foundation for rescuing silenced transgenes in transgenic cloned embryos. We investigated cytotoxic effect of the 18S RNA-Nano-flare probe on porcine fetal fibroblasts, characterized the distribution of the 18S RNA-Nano-flare probe in living cells and investigated the effect of the 18S RNA-Nano-flare probe on the development of cloned embryos after SCNT. The cytotoxic effect of the 18S RNA-Nano-flare probe on porcine fetal fibroblasts was dose-dependent, and 18S RNA was detected using the 18S RNA-Nano-flare probe. In addition, treating donor cells with 500 pM 18S RNA-Nano-flare probe did not have adverse effects on the development of SCNT embryos at the pre-implantation stage. In conclusion, we established a preliminary platform to detect RNA in live donor cells using a Nano-flare probe prior to SCNT.

Subcellular Characterization of Porcine Oocytes with Different Glucose-6-phosphate Dehydrogenase Activities.

The in vitro maturation (IVM) efficiency of porcine embryos is still low because of poor oocyte quality. Although brilliant cresyl blue positive (BCB+) oocytes with low glucose-6-phosphate dehydrogenase (G6PDH) activity have shown superior quality than BCB negative (-) oocytes with high G6PDH activity, the use of a BCB staining test before IVM is still controversial. This study aimed to shed more light on the subcellular characteristics of porcine oocytes after selection using BCB staining. We assessed germinal vesicle chromatin configuration, cortical granule (CG) migration, mitochondrial distribution, the levels of acetylated lysine 9 of histone H3 (AcH3K9) and nuclear apoptosis features to investigate the correlation between G6PDH activity and these developmentally related features. A pattern of chromatin surrounding the nucleoli was seen in 53.0% of BCB+ oocytes and 77.6% of BCB+ oocytes showed peripherally distributed CGs. After IVM, 48.7% of BCB+ oocytes had a diffused mitochondrial distribution pattern. However, there were no significant differences in the levels of AcH3K9 in the nuclei of blastocysts derived from BCB+ and BCB- oocytes; at the same time, we observed a similar incidence of apoptosis in the BCB+ and control groups. Although this study indicated that G6PDH activity in porcine oocytes was correlated with several subcellular characteristics such as germinal vesicle chromatin configuration, CG migration and mitochondrial distribution, other features such as AcH3K9 level and nuclear apoptotic features were not associated with G6PDH activity and did not validate the BCB staining test. In using this test for selecting porcine oocytes, subcellular characteristics such as the AcH3K9 level and apoptotic nuclear features should also be considered. Adding histone deacetylase inhibitors or apoptosis inhibitors into the culture medium used might improve the efficiency of IVM of BCB+ oocytes.

Evolution of mammalian and avian bornaviruses.

Recently, Avian Bornavirus (ABV) was identified to be a new member of the Bornaviridae family consisting solely of the mammal-infecting Borna disease virus (BDV). Here, to gain more insights into the evolution of these bornaviruses, the time-stamped N gene sequences of BDV genotype 1 (BDV1) and ABV were subjected to Bayesian coalescent analyses. The nucleotide substitution rates and the divergence times were estimated. Age calculations suggested that the first diversification event of the analyzed BDV1 isolates might have taken place about 300years ago, and revealed that ABV was an old virus newly recognized. Great differences were observed in the rate of nucleotide substitution and the pattern of codon usage bias between BDV1 and ABV. Moreover, the analyzed bornaviruses might be descended from an AT-rich ancestor.

[Role of microRNA in induced pluripotent stem cell].

MicroRNAs are ~22 nt long small noncoding RNA molecules that silence post-transcription gene expression. It has proven that microRNAs are widely expressed in eukaryotes and play an important role in the regulation of cell differentiation and development, growth metabolism, and many other cell activities. Induced pluripotent stem cells (iPS) are a type of pluripotent stem cells reprogrammed from somatic cells and exhibit the essential characteristics of embryonic stem cells. iPS technology has been widely applied in the biological and medical fields, and the key of it is reprogramming of somatic epigenetic state. Therefore, it is of important theoretical and practical significance to study the mechanisms of somatic reprogramming for establishment of an improved iPS technology. The methods of transfection of defined exogenetic stem factors, such as Oct4, Sox2, Klf4, and c-Myc into somatic cells through viral vectors have been continuously improving, but the genome integration and reactivation of the oncogenic gene increase the tumorigenicity of induced cells. The integration-free ways, such as adenovirus, plasmid, recombinant proteins, and L-myc replacement used in iPS technology significantly reduce the risk of cancer. However, the inducing mechanisms are still unclear. Recent studies showed that microRNA affect the process of somatic cell reprogramming, especially embryonic stem cell regulating (ESCC) family of microRNAs (miR302/367, miR200, miR-34, and miR290/295) enhances the reprogramming of embryonic fibroblasts to iPS. This article reviews the recent progresses of roles of microRNA in iPS.

Temporal and spatial expression of AIF in the mouse uterus during early pregnancy.

Apoptosis-inducing factor (AIF) is a phylogenetically old, bifunctional protein with a pro-apoptotic function and redox activity. AIF regulates apoptosis and also plays a role in the defense against stress depending on its subcellular localization. Embryo implantation is a complicated process, in which an activated blastocyst interacts with a receptive uterus. The expression and regulation of AIF were investigated in this study in the mouse uterus during early pregnancy, pseudopregnancy, delayed implantation, artificial decidualization and under hormonal treatment using in situ hybridization, immunohistochemistry and real-time PCR. During early pregnancy, temporally and spatially regulated patterns of AIF expression were found in the mouse uterus; AIF expression in the luminal epithelium and glandular epithelium is regulated by steroid hormones; AIF mRNA expression in the stroma is influenced by the active blastocyst; and AIF protein was found to be located in the cytoplasm rather than the nucleus through confocal microscope. Our data suggest that AIF might play an important role during mouse embryo implantation and that the role of AIF might be implemented through its physiological activity rather than through its pro-apoptotic function in the mouse uterus during this period.

The type and extent of injuries in vitrified mouse oocytes.

To improve the vitrification of mouse oocytes using straws, we attempted to estimate the type and extent of injuries during vitrification with a vitrification solution EAFS10/10. Injuries in oocytes were assessed based on cellular viability, the integrity of the plasma membrane, the status of the meiotic spindle/chromosomes, and morphological appearance. For morphologically normal oocytes, the ability to be fertilized and to develop into blastocysts was examined. Morphological assessment revealed 15% of oocytes to be injured by intracellular ice formed during vitrification, and 10% by osmotic swelling during removal of the cryoprotectant. When assessed by the status of spindles/chromosomes, the most sensitive criterion, damage was found in 16% of oocytes without any treatment. This value was similar to the proportion of fresh oocytes that did not cleave after insemination (13%). On exposure to EAFS10/10, the spindles/chromosomes were affected in 33% of oocytes. The exposure reduced the rate of cleavage by 18% points and the rate of development into blastocysts by 19 points. Vitrification reduced these rates by 15% and 36% points, respectively. Although the mechanism responsible for this moderate toxic effect on developmental ability is not known, information obtained in the present study will be useful to develop a practical method for the vitrification of mouse oocytes using straws.

[miRNAs play an essential role in stem cell self-renewal and differentiation].

Stem cells and miRNAs are two of the most intriguing research topics in recent years. Stem cells with properties of self-renewal and multipotency are remarkable cells since they play key roles in animal development and tumorigenesis. microRNAs (miRNAs) are approximately 22nt non-coding RNAs, which have spatiotemporally specific expression patterns, and are highly conserved between species. Researchers have demonstrated that miRNAs can regulate gene expression at post-transcriptional level, and their action is one of the essential intracellular regulatory pathways. Recent evidence showed that miRNAs can regulate stem cells self-renewal and differentiation. The evidence mostly derived from the following research strategies: (1) deletion or mutation of the genes that encoding essential enzymes for miRNAs biogenesis, such as Dicer1, Loqs, DGCR8, and Argonaute; (2) screening of specific miRNAs from stem cells and investigation of their function. Comprehension of the miRNAs functions in stem cells is of great importance for further understanding their self-renewal and differentiation mechanism, as well as identification of stem cells. This paper reviews the potential roles of miRNAs in stem cells on the basis of recent researches.

[Advances in the study of stem cell niche].

A stem cell niche is defined as the microenvironment surrounding stem cells, and it is generally composed of adjacent cells, adhensive molecules, extracellular matrix, and so on. Stem cells in different tissues differ in their niches. Stem cell niches regulate stem cell self-renewal and differentiation. According to the recent studies in stem cell niches, this review summarizes on the constitution and function of germ-line stem cells niche in fruit, hematopoietic stem cell niche, intestinal stem cell niche, hair follicle epidermal stem cell niche and neural stem cell niche in mammals, and discusses the internal mechanism of niches'action on stem cells.