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

本研究的目的是：1）构建逆转录病毒相关载体（pMX）中的肥大细胞cDNA文库; 2）发展基于逆转录病毒的表达克隆新方法; 3）应用该方法鉴定肥大细胞中与DAP 12活化亚基相关的新受体。结果如下：1）肥大细胞cDNA文库的构建用MMoLV衍生的逆转录酶从IL-3依赖的小鼠骨髓源性肥大细胞中制备双链cDNA。通过cDNA和pMX（EcoRI和Not I限制性位点）的连接，在基于逆转录病毒的质粒载体（pMX）中构建cDNA文库。该cDNA文库在E.大肠杆菌DH 10 B菌株电转化获得重组子。经过两次文库构建，我们共获得了一个包含200多万个独立克隆的肥大细胞cDNA文库。通过对肥大细胞中很少表达的mRNA种类的PCR效力来监测其完整性。We the ...更多信息 间接表达克隆法（简称间接表达克隆法）通过将DAP 12基因转染到大鼠骨髓瘤细胞系中，建立了稳定表达FLAG标记DAP 12蛋白的细胞系（12 Y 5）。当在12 Y 5中表达杀伤Ig样受体时，FLAG标记的DAP 12被分选到质膜上，所述杀伤Ig样受体已知与DAP 12缔合并且其表面表达依赖于与DAP 12的缔合。另一方面，当表达DAP 12非依赖性受体FcgRIIb时，FLAG标记的DAP 12不表达。这些结果表明，FLAG标记的DAP 12被分选到质膜依赖于DAP 12相关受体的表达。3）新的受体鉴定方法的应用我们应用新的方法鉴定了肥大细胞中与DAP 12相关的新受体。通过逆转录病毒载体法将肥大细胞cDNA文库导入12 Y 5。培养2天后，通过FACS收集FLAG表面表达阳性的细胞。然后将收集的细胞在培养物中扩增1周。将该收集和扩增循环重复三次。最后，采用有限稀释法克隆FLAG阳性细胞，并对每个克隆进行序列分析。通过4次克隆分离，获得4个独立克隆。然而，在它们之间没有鉴定出显著的序列。该方法存在的主要问题是假阳性克隆在长期培养筛选过程中易被优先扩增。少

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