[Show abstract][Hide abstract] ABSTRACT: Induced pluripotent stem cells (iPSC) are a promising cell source for cartilage regenerative medicine; however, the methods for chondrocyte induction from iPSC are currently developing and not yet sufficient for clinical application. Here, we report the establishment of a fluorescent indicator system for monitoring chondrogenic differentiation from iPSC to simplify screening for effective factors that induce chondrocytes from iPSC. We generated iPSC from embryonic fibroblasts of Col2a1-EGFP transgenic mice by retroviral transduction of Oct4, Sox2, Klf4, and c-Myc. Among the 30 clones of Col2a1-EGFP iPSC we established, two clones showed high expression levels of embryonic stem cell (ESC) marker genes, similar to control ESC. A teratoma formation assay showed that the two clones were pluripotent and differentiated into cell types from all three germ layers. The fluorescent signal was observed during chondrogenic differentiation of the two clones concomitant with the increase in chondrocyte marker expression. In conclusion, Col2a1-EGFP iPSC are useful for monitoring chondrogenic differentiation and will contribute to research in cartilage regenerative medicine.
PLoS ONE 01/2013; 8(9):e74137. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The transcription factor Klf4 has demonstrated activity in the reprogramming of somatic cells to a pluripotent state, but the molecular mechanism of this process remains unknown. It is, therefore, of great interest to understand the functional role of Klf4 and related genes in ESC regulation. Here, we show that homozygous disruption of Klf5 results in the failure of ESC derivation from ICM cells and early embryonic lethality due to an implantation defect. Klf5 KO ESCs show increased expression of several differentiation marker genes and frequent, spontaneous differentiation. Conversely, overexpression of Klf5 in ESCs suppressed the expression of differentiation marker genes and maintained pluripotency in the absence of LIF. Our results also suggest that Klf5 regulates ESC proliferation by promoting phosphorylation of Akt1 via induction of Tcl1. These results, therefore, provide new insights into the functional and mechanistic role of Klf5 in regulation of pluripotency.
[Show abstract][Hide abstract] ABSTRACT: Rat BTEB2 protein is a transcription factor with three zinc fingers that binds to GC box, and is expressed in the placenta, intestine, and testis. To understand mechanisms of gene expression of BTEB2, we have cloned the rat BTEB2 gene from a rat liver genomic library and determined the gene structure. The BTEB2 gene contained 4 exons. In the process of cloning of the BTEB2 gene, we cloned two pseudogenes for BTEB2, one of which was a processed gene. The upstream region of the bona fide gene was fused to a luciferase reporter gene, and the generated BTEB2-luciferase chimeric plasmid was transiently transfected into HeLa cells that expressed endogenous BTEB2 mRNA. Significant expression of luciferase activity was observed. Deletion analysis of the promoter region of the BTEB2 gene revealed that at least three regions are important for the activity. Upon investigation of cis-acting elements in the regions, the GC box, CCAAT box and NF-1 binding site were found. As binding factors, Sp1, CBFa, and NF-1 were identified to the DNA elements by gel mobility shift assays using specific antibodies.
[Show abstract][Hide abstract] ABSTRACT: trans-Splicing is the biological reaction that generates a mature mRNA from separate strands of pre-mRNAs. Previously, we reported that the trans-splicing between the two Sp1 pre-mRNA strands produced an mRNA with the exon 3-2-3 alignment in human HepG2 cells. Here we describe the rat counterpart as well as a newly identified variant with the exon 3-3 alignment in cultured rat cells. A qualitative evaluation of such alignments in poly(A)(+) RNA-rich preparation showed that both alignments arose from trans-splicing rather than circularization of a single strand. The identification of the trans-spliced products in both rat and human raises the possibility that trans-splicing on Sp1 pre-mRNA is rather common to mammals. It was observed that the level of the trans-spliced variants varies in different rat organs.
Biochemical and Biophysical Research Communications 11/2002; 298(1):156-62. · 2.41 Impact Factor