In the postgenomic era the elucidation of the physiological function of genes has become the rate-limiting step in the quest to understand the development and function of living organisms. Gene functions cannot be determined by high-throughput methods but require analysis in the context of the entire organism. This is particularly true in the developing vertebrate nervous system. Because of its easy accessibility in the egg, the chicken embryo has been the model of choice for developmental in vivo studies. However, its usefulness has been hampered by a lack of methods for genetic manipulation. Here we describe an approach that could compensate for this disadvantage. By combining gene silencing by dsRNA (through RNA interference, RNAi) with in ovo electroporation, we developed an efficient method to induce loss of gene function in vivo during the development of the chicken CNS. This method opens new possibilities for studying gene function not only by gain-of-function but also by loss-of-function approaches and therefore represents a new tool for functional genomics.
Gert-Jan Arts and Joost Donker were inadvertently omitted from the list of authors when this report was submitted for publication. The authors regret the omission.
Reduced translation of CEBPA mRNA has been associated with increased proliferation of bronchial smooth muscle (BSM) cells of asthma patients.Here, we assessed the effect of house dust mite (HDM) extracts on the cell proliferation ([ 3 H]-thymidine incorporation), inflammation (interleukin (IL)-6 release) and upstream translation regulatory proteins of CCAAT/enhancer-binding protein (C/EBP)a in human BSM cells of healthy controls and asthmatic patients. HDM extract significantly increased IL-6 protein and proliferation of BSM cells of asthma patients only. HDM extract reduced the C/EBPa expression in BSM cells of asthma patients, which coincided with significantly increased levels of calreticulin (CRT) protein, an inhibitor of CEBPA mRNA translation. HDM extract elicited both protease-dependent and -independent responses, which were mediated via protease-activated receptor (PAR)2 and CRT, respectively.In conclusion, HDM extract reduced CEBPA mRNA translation, specifically in asthmatic BSM cells, and 1) upregulated CRT, 2) activated PAR2, and increased 3) IL-6 expression and 4) the proliferation of asthmatic BSM cells. Hence, HDM exposure contributes to inflammation and remodelling by a nonimmune cell-mediated mechanism via a direct interaction with BSM cells. These findings may potentially explain several pathological features of this disease, in particular BSM cell hyperplasia.
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