Generation of a Transgenic Zebrafish Model for Pancreatic Beta Cell Regeneration.

BACKGROUND: Diabetes is a major worldwide health problem. It is widely accepted that the beta cell mass decreases in type I diabetes (T1D). Accordingly, beta cell regeneration is a promising approach to increase the beta cell mass in T1D patients. However, the underlying mechanisms of beta cell regeneration have yet to be elucidated. One promising avenue is to create a relevant animal model to explore the underlying molecular and cellular mechanisms of beta cell regeneration. The zebrafish can be considered a model in beta cell regeneration studies because the pancreas structure and gene expression pattern are highly conserved between human and zebrafish. MATERIALS AND METHODS: In this study, the Tol2 transposase was exploited to generate a Tg(Ins:egfp-nfsB) zebrafish model that expressed a fusion protein composed of enhanced green fluorescent protein (EGFP) and nitroreductase (NTR) under control of the Ins promoter. RESULTS: Metronidazole (MTZ) treatment of Tg(ins:egfp-nfsB) zebrafish larvae led to selective ablation of beta cells. Proof-of-concept evidence for beta cell regeneration in the transgenic larvae was observed two days after withdrawal of MTZ. CONCLUSION: This study suggests that the Tg(ins:egfp-nfsB) zebrafish can be used as a disease model to study beta cell regeneration and elucidate underlying mechanisms during the regeneration process.

Establishment of a Transgenic Zebrafish Expressing GFP in the Skeletal Muscle as an Ornamental Fish.

BACKGROUND: Transgenic animals have a critical role in the advancement of our knowledge in different fields of life sciences. Along with recent advances in genome engineering technologies, a wide spectrum of techniques have been applied to produce transgenic animals. Tol2 transposase method is one of the most popular approaches that were used to generate transgenic animals. The current study was set out to produce an ornamental fish, which express enhanced green fluorescent protein (EGFP) under control of mylpfa promoter by using Tol2 transposase method. MATERIALS AND METHODS: Polymerase chain reaction (PCR) cloning method was performed to insert zebrafish myosin promoter (mylpfa) into Tol2-EGFP plasmid at the upstream of EGFP. In vitro transcription method was used to prepare the transposase mRNA. The Tol2-EGFP plasmid and transposase mRNA were then co-injected into the one-cell stage of zebrafish zygotes. After two days, the fluorescent microscopic analysis was used to select transgenic zebrafishes. RESULTS: Our data showed that the optimum concentration for recombinant Tol2 vector and transposase mRNA were 50 ng/ul and 100 ng/ul, respectively. The results also revealed that the quality of embryos and quantity of injected construct had the important effects on Tol2 transposase method efficiency. CONCLUSION: Data showed that Tol2 transposase is an appropriate method to generate zebrafish transgene. Our finding also showed that mylpfa promoter is a strong promoter that can be used as a selected promoter in the ornamental fish industry.

Temporal Gene Expression and DNA Methylation during Embryonic Stem Cell Derivation.

OBJECTIVE: Dual inhibition of mitogen-activated protein kinase (MAPK) kinase (also known as MEK) and transforming growth factor ß (TGFß) type I receptors by PD0325901 and SB431542, known as R2i has been introduced as a highly efficient approach to the generation of mouse embryonic stem cells (ESC). In the present study, we investigated the molecular mechanisms underlying ESC derivation in the R2i condition. MATERIALS AND METHODS: In this experimental study, zona-free whole E3.5 blastocysts were seeded on mouse embryonic fibroblast (MEF) feeder cells in both R2i and serum conventional media. The isolated inner cell mass (ICM), ESCs and the ICM-outgrowths were collected on days 3, 5 and 7 post-blastocyst culture for quantitative real timepolymerase chain reaction (qRT-PCR) analysis as well as to assess the DNA methylation status at the time points during the transition from ICM to ESC. RESULTS: qRT-PCR revealed a significantly higher expression of the pluripotency-related genes (Oct4, Nanog, Sox2, Rex1, Dppa3, Tcf3, Utf1, Nodal, Dax1, Sall4 and ß-Catenin) and lower expression of early differentiation genes (Gata6, Lefty2 and Cdx2) in R2i condition compared to the serum condition. Moreover, the upstream region of Oct4 and Nanog showed a progressive increase in methylation levels in the upstream regions of the genes following in R2i or serum conditions, followed by a decrease of DNA methylation in ESCs obtained under R2i. However, the methylation level of ICM outgrowths in the serum condition was much higher than R2i, at levels that could have a repressive effect and therefore explain the absence of expression of these two genes in the serum condition. CONCLUSION: Our investigation revealed that generation of ESCs in the ground-state of pluripotency could be achieved by inhibiting the MEK and TGF-ß signaling pathways in the first 5 days of ESC derivation.

Derivation of Pluripotent Cells from Mouse SSCs Seems to Be Age Dependent.

Here, we aimed to answer important and fundamental questions in germ cell biology with special focus on the age of the male donor cells and the possibility to generate embryonic stem cell- (ESC-) like cells. While it is believed that spermatogonial stem cells (SSCs) and truly pluripotent ESC-like cells can be isolated from adult mice, it remained unknown if the spontaneous conversion of SSCs to ESC-like cells fails at some age. Similarly, there have been differences in the literature about the duration of cultures during which ESC-like cells may appear. We demonstrate the possibility to derive ESC-like cells from SSC cultures until they reach adolescence or up to 7 weeks of age, but we point out the impossibility to derive these cells from older, mature adult mice. The inability of real adult SSCs to shift to a pluripotent state coincides with a decline in expression of the core pluripotency genes Oct4, Nanog, and Sox2 in SSCs with age. At the same time genes of the spermatogonial differentiation pathway increase. The generated ESC-like cells were similar to ESCs and express pluripotency markers. In vitro they differentiate into all three germ lineages; they form complex teratomas after transplantation in SCID mice and produce chimeric mice.

Suppression of transforming growth factor ß signaling promotes ground state pluripotency from single blastomeres.

STUDY QUESTION: Can transforming growth factor ß (TGFß) inhibition promote ground state pluripotency of embryonic stem cells (ESCs) from single blastomeres (SBs) of cleavage embryos in different mouse stains? SUMMARY ANSWER: Small molecule suppression of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) and TGFß signaling (designated as R2i) can enhance the generation of mouse ESCs from SBs of different cleavage stage embryos compared with the dual suppression of ERK1/2 and glycogen synthase kinase 3 (GSK3), designated as 2i, regardless of the strain of mouce. WHAT IS KNOWN ALREADY: It is known that chemical inhibition of TGFß promotes ground state pluripotency in the generation and sustenance of naïve ES cells from mouse blastocysts compared with the well-known 2i condition. However, the positive effect of this inhibition on mouse ESCs from early embryonic SBs remains obscure. STUDY DESIGN, SIZE, DURATION: We used 155 cleavage-stage mouse embryos to optimize the culture conditions for blastocyst development. Then, to assess the effects of R2i and 2i on ESC generation from SBs, we cultured isolated SBs in 2i and R2i for 10 days. SBs were replated under the same conditions to produce ESCs. In total, 46 embryos and 321 SBs from two- to eight-cell stages were recovered from NMRI and BALB/c mouse strains and used in this study. PARTICIPANTS/MATERIALS, SETTING, METHODS: Blastomeres from 2- to 8-cell stage mouse embryos were dispersed and individually seeded into a 96-well plates that included mitotically inactivated feeder cells. ESCs were generated in B27N2 defined medium supplemented with R2i or 2i. Randomly selected ESC lines, generated from SBs of each stage, were assessed for pluripotency and germ-line transmission. MAIN RESULTS AND THE ROLE OF CHANCE: We demonstrated that dual inhibition of ERK1/2 and TGFß (R2i) enhanced efficient blastocyst development and efficient establishment of ESCs from SB of 2- to 8-cell stage mouse embryos compared with the dual inhibition of ERK1/2 and GSK3 (2i) regardless of the embryonic stage and strain of mice. The proportions of SBs that produced ESC were 50-60 versus 20-30%. LIMITATIONS, REASONS FOR CAUTION: This study was done with mouse embryos, it is not known whether these findings are transferable to humans. WIDER IMPLICATIONS OF THE FINDINGS: These findings resulted in an increased efficiency of ESC generation from one biopsied blastomere for autogeneic or allogeneic matched pluripotent cells without the need to destroy viable embryos. The results also provided information about the developmental capacity of early embryonic blastomeres. STUDY FUNDING/COMPETING INTERESTS: This study was funded by grants provided from Royan Institute, the Iranian Council of Stem Cell Research and Technology and the Iran National Science Foundation. The authors have no conflict of interest to declare.

Efficient induction of pluripotency in primordial germ cells by dual inhibition of TGF-ß and ERK signaling pathways.

Primordial germ cells (PGCs) have the ability to be reprogrammed into a pluripotent state and are defined as embryonic germ cells (EGCs) in vitro. EGC formation is more efficient, has a shorter culture period than somatic cell reprogramming, and does not require exogenous genetic manipulation. Therefore, EGCs are a good model to analyze mechanisms by which committed cells acquire a pluripotent state. In the present study we have attempted to elucidate a more defined and robust protocol that promotes EGC generation through the suppression of transforming growth factor-ß (TGF-ß) and extracellular signal-regulated kinase (ERK) signaling pathways by SB431542 (SB) and PD0325901 (PD), respectively. Under this condition the efficiency of transformation of PGCs into EGCs was more than the inhibition of glycogen synthase kinase 3 and ERK signaling pathways. Pluripotency of the resultant-derived EGC lines were further confirmed by gene expression, immunofluorescent staining, directed differentiation ability, teratoma formation, and their contribution to chimeric mice and germ-line transmission. These results showed that PGCs from different embryonic stages (E8.5 and E12.5) could be reprogrammed, maintained, and expanded efficiently under feeder- and serum-free chemically defined conditions by dual inhibition of TGF-ß and ERK signaling pathways, regardless of the animal's genetic background.