A visible phagemid system for the estimation of Cre-mediated recombination efficiency

Diversity in phage antibody libraries is important for obtaining useful high-affinity antibodies. The Cre-lox system is generally employed to increase the size of phage antibody libraries. However, estimation of library sizes after Cre-mediated recombination is difficult, since time-consuming nucleo...

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Bibliographic Details
Published in:Journal of immunological methods 2003-09, Vol.280 (1), p.165-173
Main Authors: Kwon, Myung-Hee, Lee, Myung-Shin, Hong, Seung-Ho, Kim, Kyongmin Hwang, Shin, Ho-Joon, Park, Sun, Lee, Chi-Hyung, Kim, Hyung-Il
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Antibodies, immunoglobulins
Antibody library size
Base Sequence
Biological and medical sciences
Cre-mediated recombination
DNA, Recombinant - genetics
Fundamental and applied biological sciences. Psychology
Fundamental immunology
Genes, Immunoglobulin
Genetic Vectors
Immunoglobulin Heavy Chains - genetics
Immunoglobulin Light Chains - genetics
Immunoglobulin Variable Region - genetics
Integrases - genetics
Mice
Molecular immunology
Monoclonal antibodies
Peptide Library
Recombination efficiency
Recombination, Genetic
Viral Proteins - genetics
Visible phagemid system
X-gal staining
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Summary:Diversity in phage antibody libraries is important for obtaining useful high-affinity antibodies. The Cre-lox system is generally employed to increase the size of phage antibody libraries. However, estimation of library sizes after Cre-mediated recombination is difficult, since time-consuming nucleotide sequence analyses are required. We have developed a visible phagemid vector system that facilitates the estimation of recombination efficiency between V H and V L genes. In this system, intermolecular recombination between V L genes flanked by incompatible lox sites is coincident with the expression of functional β-galactosidase. Recombination efficiency can be calculated simply by counting the number of blue colonies on X-gal-containing medium. Molecular analyses of plasmids isolated from blue colonies reveal a novel V H/V L combination. Our results suggest that this newly developed visible phagemid system may be reliably used for the measurement of recombination efficiency, which would enable precise evaluation of the diversity of phage antibody libraries.
ISSN:0022-1759
1872-7905
DOI:10.1016/S0022-1759(03)00261-8