Establishment of Germ-line Competent C57BL/6J Embryonic Stem Cell Lines

Objective To establish C57BL/6J embryonic stem （ES） cell lines with potential germ- line contribution Methods ES cells were isolated from blastocyst inner cell mass of C5 7BL/6J mice, and cultured for 15 passages, and then injected into blastococels of ICR mice blastocysts to establish chimeric mice. Results Three ES cell lines （mC57ES1,mC57ES3, mC57ES7） derived from the inner cell mass of C57BL/6J mice blastocysts were established. They were characteristic of undifferentiated state, including normal XY karyotype, expression of a specific cell surface marker “stage-specific embryonic antigen-I” and alkaline phosphatase in continuous passage. When injected into immunodeficient mice, mC57ES1 cells consistently differentiated into derivatives of all three embryonic germ layers. When mC57ES1 cells were transferred into ICR mice blastocysts, 4 chimeric mice have been obtained. One male of them revealed successful germ-line transmission. Conclussion We have obtained C57BL/6J ES cell lines with a potential germ-line contribution, which can be used to generate transgenic and gene knock-out mice.

Embryoid bodies formation and differentiation from mouse embryonic stem cells in collagen/Matrigel scaffolds

Embryonic stem （ES） cells have the potential to develop into any type of tissue and are considered as a promising source of seeding cells for tissue engineering and transplantation therapy.The main catalyst for ES cells differentiation is the growth into embryoid bodies （EBs）,which are utilized widely as the trigger of in vitro differentiation.In this study,a novel method for generating EBs from mouse ES cells through culture in collagen/Matrigel scaffolds was successfully established.When single ES cells were seeded in three dimensional collagen/Matrigel scaffolds,they grew into aggregates gradually and formed simple EBs with circular structures.After 7 days＇ culture,they formed into cystic EBs that would eventually differentiate into the three embryonic germ layers.Evaluation of the EBs in terms of morphology and potential to differentiate indicated that they were typical in structure and could generate various cell types;they were also able to form into tissue-like structures.Moreover,with introduction of ascorbic acid,ES cells differentiated into cardiomyocytes efficiently and started contracting synchronously at day 19.The results demonstrated that collagen/Matrigel scaffolds supported EBs formation and their subsequent differentiation in a single three dimensional environment.

Regulation beyond genome sequences: DNA and histone methylation in embryonic stem cells

Embryonic stem(ES)cells distinct themselves from other cell type populations by their pluripotent ability.The unique features of ES cells are controlled by both genetic and epigenetic factors.Studies have shown that the methylation status of DNA and histones in ES cells is quite different from that of differentiated cells and somatic stem cells.Herein,we summarized recent advances in DNA and histone methylation studies of mammalian ES cells.The methylation status of several key pluripotent regulatory genes is also discussed.

Rapid and Cost-Effective Gene Targeting in Rat Embryonic Stem Cells by TALENs

The rat is the preferred animal model in many areas of biomedical research and drug development. Genetic manipulation in rats has lagged behind that in mice due to the lack of efficient gene targeting tools. Previously, we generated a knockout rat via conventional homologous recombination in rat embryonic stern （ES） cells. Here, we show that efficient gene targeting in rat ES cells can be achieved quickly through transcription activator-like effector nuclease （TALEN）-mediated DNA double-strand breaks. Using the Golden Gate cloning technique, we constructed a pair of TALEN targeting vectors for the gene of interest in 5 days. After gene transfection, the targeted rat ES cell colonies were isolated, screened, and confirmed by PCR without the need of drug selection. Our results suggest that TALEN-mediated gene targeting is a superior means of establishing genetically modified rat ES cell lines with high efficiency and short turnaround time.

Establishment of novel embryonic stem(ES) cell lines from OG2/rtTA blastocysts

Embryonic stem （ES） cells derived from the pre-implantation blastocyst-stage embryos have been widely used to investigate the molecular events determining pluripotency and cell lineage differentiation. As the first discovered ES-specific transcription factor, Oct4 has been considered as the core pluripotency factor of ES cells. In the present study, we successfully established seven ES lines from the blastocysts collected from female OG2 （Oct4-GFP transgenic） mice, which have been crossed with male rtTA transgenic mice. The pluripotency of the ES cell lines can be visualized by the expression of Oct4-GFP under fluorescent microscopy and germ-line transmission capability has been further confirmed. More importantly, the presence of rtTA could induce transgene＇s expression with the help of doxycycline. Therefore, these ES cell lines provide an excellent tool to further discover novel factors affecting pluripotency and to investigate the molecular mechanism of reprogramming in defined transcription factors mediated nuclear reprogramming.

Subcellular redistribution and sequential recruitment of macromolecular components during SGIV assembly

Virus infection consists of entry, synthesis of macro- molecular components, virus assembly and release. Understanding of the mechanisms underlying each event is necessary for the intervention of virus infection in human healthcare and agriculture. Here we report the visualization of Singapore grouper iridovirus （SGIV） assembly in the medaka haploid embryonic stem （ES） cell line HX1. SGIV is a highly infectious DNA virus that causes a massive loss in marine aquaculture. Ectopic expression of VP88GFP, a fusion between green fluo- rescent protein and the envelope protein VP088, did not compromise the ES cell properties and susceptibility to SGIV infection. Although VP88GFP disperses evenly in the cytoplasm of non-infected cells, it undergoes aggregation and redistribution in SGIV-infected cells. Real-time visualization revealed multiple key stages of VP88GFP redistribution and the dynamics of viral assembly site （VAS）. Specifically, VP88GFP entry into and condensation in the VAS occurred within a 6-h duration, a similar duration was observed also for the release of VP88GFP-containing SGIV out of the cell, Taken together, VP088 is an excellent marker for visu- alizing the SGIV infection process. Our results provide new insight into macromolecular component recruit- ment and SGIV assembly.