Lethality-based selection of recombinant genes in mammalian cells: application to identifying tumor antigens

Area of Science:

• Molecular Biology
• Immunology
• Virology

Background:

• Mammalian expression vectors are crucial for constructing cDNA libraries, but recovery is limited to actively dividing cells.
• This limitation hinders the selection of genes involved in cell death or growth arrest, such as those encoding cytotoxic T-cell antigens or pro-apoptotic factors.
• Existing methods are inefficient for isolating genes from non-proliferating cells.

Purpose of the Study:

• To develop a novel mammalian expression vector system for constructing cDNA libraries from non-dividing cells.
• To overcome the limitations of traditional vectors in selecting genes related to cell lethality or growth arrest.
• To enable the efficient recovery of functional genes from cells that have undergone selection-based cell death.

Main Methods:

• Construction of representative cDNA libraries using a poxvirus-based vector system.
• Application of lethality-based selection to recover desired genes from non-dividing cells.
• Utilizing the poxvirus vector for efficient gene recovery irrespective of host cell proliferation.

Main Results:

• Successfully constructed cDNA libraries in a poxvirus-based vector that can be recovered from cells undergoing lethality-based selection.
• Demonstrated the feasibility of selecting genes from non-proliferating cells.
• Applied the strategy to successfully select a gene encoding a cytotoxic T-cell target antigen found in multiple tumors.

Conclusions:

• Poxvirus-based expression vectors provide a robust platform for creating and recovering cDNA libraries from non-dividing mammalian cells.
• This novel approach significantly enhances the efficiency of selecting genes that mediate cell death or growth arrest.
• The strategy has direct applications in identifying key genes, such as tumor antigens, relevant to cancer immunology and therapy.