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A powerful new technique for isolating genes encoding cell surface antigens using retroviral expression cloning
A C Zannettino1, J R Rayner, L K Ashman
1Hanson Centre for Cancer Research, Matthew Roberts Laboratory, Division of Haematology, Adelaide, South Australia, Australia.
Journal of Immunology (Baltimore, Md. : 1950)
|January 15, 1996
Summary
This study introduces a new method for quickly isolating genes for cell surface molecules (CSM) using retroviral cDNA libraries. This technique efficiently identifies genes for CSM, advancing gene discovery and understanding of cell surface proteins.
Area of Science:
- Molecular Biology
- Immunology
- Genetics
Background:
- Retroviral vectors enable stable gene introduction into cells, surpassing transfection efficiencies.
- They facilitate the expression of complex cDNA libraries across diverse cell types, including hematopoietic cells.
Purpose of the Study:
- To develop a novel method for rapidly isolating genes encoding cell surface molecules (CSM).
- To utilize a human bone marrow stromal cell cDNA library for expression cloning of CSM.
Main Methods:
- Construction of a cDNA library in the pRUFneo retroviral vector.
- Implementation of a highly efficient selection strategy using monoclonal antibodies (mAb) and antibody-coated magnetic beads.
- Application of this method to isolate genes from a human bone marrow stromal cell library.
Main Results:
- Successfully isolated six cDNAs encoding previously defined CSM, including beta 1 integrin and endoglin.
- Identified the genes and corresponding CSM for three previously unclustered mAbs.
- Demonstrated the utility of retroviral cDNA libraries for expression cloning of CSM.
Conclusions:
- The novel method provides a rapid and efficient approach for isolating genes encoding CSM.
- Retroviral cDNA libraries are versatile tools for expression cloning of cell surface molecules.
- This technique advances the identification and characterization of novel CSM.