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Identification of immature podocyte specific antigen using retrovirus-mediated gene transfer and cell sorting

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Abstract

Background

For identifying an antigen recognizable by a monoclonal antibody (mAb), the cDNA cloning method using expression cDNA library has become increasingly popular. For analysis of the mAb clone 2E3, which recognizes the cell membrane surface of immature podocytes, we developed an expression cloning strategy that identified the targeted antigen using retrovirus-mediated gene transfer and enrichment by cell sorting.

Methods

In this experiment, the NIH3T3 cell line was infected by a retrovirally amplified cDNA library derived from the same fetal murine kidneys that provided the immunized antigen. Infected NIH3T3 staining for mAb clone 2E3 was concentrated by cell sorting, followed by identification of the integrated gene.

Results

The infected cells reacted with mAb were highly enriched by two rounds of cell sorting. As a result of sequencing the inserted gene from the enriched cells, we isolated the low-affinity nerve growth factor receptor (NGFR) gene. Furthermore, the NIH3T3 cell line that was enforced by recovery of NGFR cDNA reacted with mAb clone 2E3.

Conclusions

Monoclonal antibodies recognizing cell membrane surface molecules are essential tools for immunohistochemistry and flow cytometry analysis, and the application of a combination of retrovirus-mediated gene transfer and cell sorting is beneficial for identifying the targeted antigen.

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Authors and Affiliations

  1. Laboratory of Stem Cell Therapy, Center for Experimental Medicine, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan

    Joichi Usui, Mitsujiro Osawa, Satoshi Yamazaki, Yohei Morita & Hiromitsu Nakauchi

  2. Majors of Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, 305-3525, Japan

    Joichi Usui & Akio Koyama

Authors
  1. Joichi Usui
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  2. Mitsujiro Osawa
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  3. Satoshi Yamazaki
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  4. Yohei Morita
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  5. Akio Koyama
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  6. Hiromitsu Nakauchi
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Corresponding author

Correspondence to Hiromitsu Nakauchi.

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Usui, J., Osawa, M., Yamazaki, S. et al. Identification of immature podocyte specific antigen using retrovirus-mediated gene transfer and cell sorting. Clin Exp Nephrol 9, 292–296 (2005). https://doi.org/10.1007/s10157-005-0389-0

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  • DOI: https://doi.org/10.1007/s10157-005-0389-0

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