| Embryonic stem cells (ESC) are cells generated from inner cell mass (ICM) of pre-implantation embryos. They can be propagated stably in vitro for a long time, and preserve the capacity of differentiating into all cell lineages derived from the three germ layers, which still keep normal diploid karyotype. ESCs are excellent source for cell therapy, tissue engineering and drug screening. They are also superior model for research in embryo development at the early period.ESCs are different from other cells because of their characterizations of highly self-renew ability and differentiation pluripotency. ESC specific markers play an important role in characterization or isolation of ESC lines and elucidation for the mechanism of self-renewal or differentiation in ESC. Especially cell surface markers, which are involved in extracellular signal transduction or cell communication, are of great interest. However, the reported ESC markers are still limited, which are co-expressed with the other adult stem cells or malignant cells. More ESC markers are still to be found. Yet it is not an easy work for identification of ESC markers: ESC markers, especially surface markers, are of low abundance, complicated modification and quick variation; complicated system of ESC is not suitable for large scale culture; the heterogeneous population in ESC also increases the difficulty.Presently approaches to screen for ESC markers include monoclonal antibody (mAb) technique, transcriptomics technique and proteomics technique. In this research we use mouse ESC to immunize rabbit, and screen for mAbs which can specifically bind to ESC. By combining Immunoprecipitation with Mass Spectrometry, it is possible to identify novel markers for ESCs. Frequently used ESC markers (SSEA-1, SSEA-3, TRA-1-60, etc.) were not derived from ESC directly and their host for immunization was mostly mouse. Comparing with mouse mAbs, rabbit mAbs have many advantages: first, rabbit are more sensitive to exogenous immunogens, producing antibodies to mouse immunogens or recognize different epitopes from mice; second, rabbit antibodies have a higher affinity; third, rabbit have a larger spleen, which could produce more spleen cells for generating hybridomas and selecting different antibodies. Large quantity of rabbit mAbs can be produced efficiently using ES cells as immunogen. Hence, it is more likely to screen mAbs against novel markers of ES cells from those rabbit mAbs.In this study, we generated more than 200 hybridomas secreting ES cell binding antibodies, 20 hybridomas were elected for subclone and 14 stable hybridomas were obtained. Characterization of these mAbs shows that most of they could specifically bind to ESCs, and the antigens of them were partly expressed in adult tissues. Study for one hybridoma 9# shows that: it secretes mAb against inositol 1,4,5-triphosphate receptor, which is expressed in endoplasmic reticulum, and regulates the release of Ca2+ from the endoplasmic reticulum (ER). The other mAbs are still in study until now.
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