ABSTRACT
Activation-induced cytidine deaminase (AID) is required for the generation of antibody diversity through initiating both somatic hypermutation (SHM) and class switch recombination. A few research groups have successfully used the feature of AID for generating mutant libraries in directed evolution of target proteins in B cells in vitro. B cells, cultured in suspension, are not convenient for transfection and cloning. In this study, we established an AID-based mutant accumulation and sorting system in adherent human cells. Mouse AID gene was first transfected into the human non-small cell lung carcinoma H1299 cells, and a stable cell clone (H1299-AID) was selected. Afterwards, anti-hTNF-α scFv (ATscFv) was transfected into H1299-AID cells and ATscFv was displayed on the surface of H1299-AID cells. By 4-round amplification/flow cytometric sorting for cells with the highest affinities to hTNF-alpha, two ATscFv mutant gene clones were isolated. Compared with the wild type ATscFv, the two mutants were much more efficient in neutralizing cytotoxicity of hTNF-alpha. The results indicate that directed evolution by somatic hypermutation can be carried out in adherent non-B cells, which makes directed evolution in mammalian cells easier and more efficient.
Subject(s)
Animals , Humans , Mice , Antibody Affinity , Cells, Cultured , Cytidine Deaminase , Genetics , Metabolism , HEK293 Cells , Immunoglobulin Variable Region , Genetics , Allergy and Immunology , Mutation , Single-Chain Antibodies , Chemistry , Genetics , Allergy and Immunology , Somatic Hypermutation, Immunoglobulin , Genetics , Allergy and Immunology , Tumor Necrosis Factor-alpha , Allergy and ImmunologyABSTRACT
The Rad1 gene is evolutionarily conserved from yeast to human. The fission yeast Schizosaccharomyces pombe Rad1 ortholog promotes cell survival against DNA damage and is required for G(2)/M checkpoint activation. In this study, mouse embryonic stem (ES) cells with a targeted deletion of Mrad1, the mouse ortholog of this gene, were created to evaluate its function in mammalian cells. Mrad1 (-/-) ES cells were highly sensitive to ultraviolet-light (UV light), hydroxyurea (HU) and gamma rays, and were defective in G(2)/M as well as S/M checkpoints. These data indicate that Mrad1 is required for repairing DNA lesions induced by UV-light, HU and gamma rays, and for mediating G(2)/M and S/M checkpoint controls. We further demonstrated that Mrad1 plays an important role in homologous recombination repair (HRR) in ES cells, but a minor HRR role in differentiated mouse cells.
Subject(s)
Animals , Mice , Cell Division , Cell Proliferation , DNA Damage , DNA Repair , Embryonic Stem Cells , Metabolism , Exonucleases , Genetics , Metabolism , Physiology , G2 Phase , Gamma Rays , Gene Deletion , Hydroxyurea , Pharmacology , Ultraviolet RaysABSTRACT
Fluorescence resonance energy transfer (FRET) is the energy transfer from an activated donor fluorophore to a receiving fluorophore. The efficiency of the energy transfer is the function of the distance between the two fluorophores, and is very sensitive to the distance. Its effective response distance is between 1~10 nm, thus it can be used to measure the distance between atoms or molecules. The feature of FRET is very useful in researches on conformational changes, interaction between macromolecules and signal transductions within live cells, and FRET has become a powerful tool in biomedical investigations. However, biological processes often involve interactions between more than two macromolecules, and FRET using two color fluorophores cannot meet the research demand. Recently, two research groups made breakthrough, establishing a novel FRET technique using three color fluorophores based on confocol microscopy and flow cytometry, respectively. The invention will significantly advance researches in biological and related fields.
ABSTRACT
Cell cycle checkpoints are protective mechanism responding to DNA damages originated from externalor internal factors. When cells are exposed to genotoxic stress or when nutrition crisis occurs, cell cycleprogression is usually stopped or slowed down by cell cycle checkpoints to allow for DNA repair or for handlingthe crisis. Besides, recent studies suggest that some cell cycle checkpoint proteins are also involved in regulatingphysiological DNA replication via controlling the rate of DNA replication. Cell cycle checkpoint proteins ATR,9-1-1 complex, Chk1, Cdc25A and CDK2 may participate in this process. This kind of regulation is supposed to bevery important for ensuring accurate DNA replication and maintaining genomic stability.