Different cis-regulatory DNA elements mediate developmental stage- and tissue-specific expression of the human COL2A1 gene in transgenic mice

Expression of the type II collagen gene (human COL2A1, mouse Col2a1) heralds the differentiation of chondrocytes. It is also expressed in progenitor cells of some nonchondrogenic tissues during embryogenesis. DNA sequences in the 5' flanking region and intron 1 are known to control tissue- specific expression in vitro, but the regulation of COL2A1 expression in vivo is not clearly understood. We have tested the regulatory activity of DNA sequences from COL2A1 on the expression of a lacZ reporter gene in transgenic mice. We have found that type II collagen characteristic expression of the transgene requires the enhancer activity of a 309-bp fragment (+2,388 to +2,696) in intron 1 in conjunction with 6.1-kb 5' sequences. Different regulatory elements were found in the 1.6-kb region (+701 to +2,387) of intron 1 which only needs 90-bp 5' sequences for tissue-specific expression in different components of the developing cartilaginous skeleton. Distinct positive and negative regulatory elements act together to control tissue- specific transgene expression in the developing midbrain neuroepithelium. Positive elements affecting expression in the midbrain were found in the region from -90 to -1,500 and from +701 to +2,387, whereas negatively acting elements were detected in the regions from -1,500 to -6,100 and +2,388 to +2,855.published_or_final_versio

Human COL2A1-directed SV40 T antigen expression in transgenic and chimeric mice results in abnormal skeletal development

The ability of SV40 T antigen to cause abnormalities in cartilage development in transgenic mice and chimeras has been tested. The cis- regulatory elements of the COL2A1 gene were used to target expression of SV40 T antigen to differentiating chondrocytes in transgenic mice and chimeras derived from embryonal stem (ES) cells bearing the same transgene. The major phenotypic consequences of transgenic (pAL21) expression are malformed skeleton, disproportionate dwarfism, and perinatal/neonatal death. Expression of T antigen was tissue specific and in the main characteristic of the mouse α1(II) collagen gene. Chondrocyte densities and levels of α1(II) collagen mRNAs were reduced in the transgenic mice. Islands of cells which express cartilage characteristic genes such as type IIB procollagen, long form α1(IX) collagen, α2(XI) collagen, and aggrecan were found in the articular and growth cartilages of pAL21 chimeric fetuses and neonates. But these cells, which were expressing T antigen, were not properly organized into columns of proliferating chondrocytes. Levels of α1(II) collagen mRNA were reduced in these chondrocytes. In addition, these cells did not express type X collagen, a marker for hypertrophic chondrocytes. The skeletal abnormality in pAL21 mice may therefore be due to a retardation of chondrocyte maturation or an impaired ability of chondrocytes to complete terminal differentiation and an associated paucity of some cartilage matrix components.published_or_final_versio

Reprogramming of Mouse Calvarial Osteoblasts into Induced Pluripotent Stem Cells

Previous studies have demonstrated the ability of reprogramming endochondral bone into induced pluripotent stem (iPS) cells, but whether similar phenomenon occurs in intramembranous bone remains to be determined. Here we adopted fluorescence-activated cell sorting-based strategy to isolate homogenous population of intramembranous calvarial osteoblasts from newborn transgenic mice carrying both Osx1-GFP::Cre and Oct4-EGFP transgenes. Following retroviral transduction of Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc), enriched population of osteoblasts underwent silencing of Osx1-GFP::Cre expression at early stage of reprogramming followed by late activation of Oct4-EGFP expression in the resulting iPS cells. These osteoblast-derived iPS cells exhibited gene expression profiles akin to embryonic stem cells and were pluripotent as demonstrated by their ability to form teratomas comprising tissues from all germ layers and also contribute to tail tissue in chimera embryos. These data demonstrate that iPS cells can be generated from intramembranous osteoblasts.published_or_final_versio

Knowledge and Attitudes Toward Brain Stem Death Among University Undergraduates

Background: Withdrawal of life support and organ procurement for transplantation are the main implications of a diagnosis of brain stem death (BSD). Various factors may impact this important decision-making process. The present study sought to investigate the knowledge and attitudes about BSD among university undergraduates as a "well-informed" subgroup of our local population. Methods: A cross-sectional questionnaire survey was administered to a sample of nonmedical university undergraduate students in Hong Kong. Results: The subjects' overall knowledge of BSD was unsatisfactory. Only 24% of subjects knew that BSD was the equivalent of legal death in Hong Kong. Among subjects who agreed to withdraw life support treatment from themselves upon the diagnosis of BSD, 30% and 24% refused to do so for their family members or a stranger, respectively. Subjects who agreed to withdraw life support showed significantly better knowledge about BSD than did those who did not agree. Concerns about doctors' inclination to diagnose BSD to save resources and extract organs for transplantation were not observed to negatively affect subjects' decisions about life support withdrawal. Conclusion: The level of knowledge is an important factor affecting an individual's decision concerning withdrawal of life support therapy upon the diagnosis of BSD. Adequate explanation and counseling are important to facilitate family members in coping with this important end-of-life issue. © 2009 Elsevier Inc. All rights reserved.link_to_subscribed_fulltex

Knowledge and Attitudes Toward Brain Stem Death Among University Undergraduates

Background: Withdrawal of life support and organ procurement for transplantation are the main implications of a diagnosis of brain stem death (BSD). Various factors may impact this important decision-making process. The present study sought to investigate the knowledge and attitudes about BSD among university undergraduates as a "well-informed" subgroup of our local population. Methods: A cross-sectional questionnaire survey was administered to a sample of nonmedical university undergraduate students in Hong Kong. Results: The subjects' overall knowledge of BSD was unsatisfactory. Only 24% of subjects knew that BSD was the equivalent of legal death in Hong Kong. Among subjects who agreed to withdraw life support treatment from themselves upon the diagnosis of BSD, 30% and 24% refused to do so for their family members or a stranger, respectively. Subjects who agreed to withdraw life support showed significantly better knowledge about BSD than did those who did not agree. Concerns about doctors' inclination to diagnose BSD to save resources and extract organs for transplantation were not observed to negatively affect subjects' decisions about life support withdrawal. Conclusion: The level of knowledge is an important factor affecting an individual's decision concerning withdrawal of life support therapy upon the diagnosis of BSD. Adequate explanation and counseling are important to facilitate family members in coping with this important end-of-life issue. © 2009 Elsevier Inc. All rights reserved.link_to_subscribed_fulltex