Development of new cloning method and its application for novel receptor identification in mast cells

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Objectives of this study are 1) construction of mast cell cDNA library in retrovirus related vector (pMX), 2) development of new method for retrovirus-based expression cloning and 3) its application for identification of novel receptor associated with DAP12 activation subunit in mast cell. The results are summarized in the following three sections.1) Construction of mast cell cDNA libraryThe double stranded cDNA was prepared from bone marrow-derived IL-3-dependent murine mast cells by MMoLV-derived reverse transcriptase. The cDNA library was constructed in retrovirus-based plasmid vector (pMX) by ligation of cDNA and pMX (EcoRI and Not I restriction sites). The cDNA library was once amplified in E. coli (DH10B strain) by electro-transformation. After performing library constructions twice, we totally obtained a mast cell cDNA library containing more than 2 millions independent clones. Its integrity was monitored by PCR efficacy for the mRNA species rarely expressed in mast cell. We the … More reby concluded that our cDNA library was of good complexity to take advantage in further study.2) Indirect expression cloning method (mentioned as new method)We established a stable cell line (12Y5) expressing FLAG-tagged DAP12 protein in rat myeloma cell line by its relevant DNA transfection. When expressed the killer Ig-like receptor in 12Y5, which is known to associate with DAP12 and whose surface expression is dependent on association with DAP12, FLAG-tagged DAP12 was sorted to plasma membrane. On the other hand, when expressed the DAP12-independent receptor, FcgRIIb, FLAG-tagged DAP12 was not. These findings indicate that FLAG-tagged DAP12 is sorted to plasma membrane as depending on expression of DAP 12-associating receptor.3) Application of new method for identification of novel receptorWe applied the new method to identify novel receptor associating with DAP12 in mast cell. The mast cell cDNA library was introduced into 12Y5 by retrovirus-based method. After culturing for 2 days, cells positive for surface expression of FLAG were collected by FACS. Then the collected cells were expanded in culture for 1 week. This collection and expansion cycle was repeated three times. Finally, the FLAG-positive cells were cloned by limiting dilution method, each clone was subjected to sequence analysis. We isolated 4 independent clones through 4 times cloning performances. However no significant sequence was identified among them. As the main problem of this method, we concluded that false-positive clones could preferentially be amplified on the way of long-term culture for selection. Less