Targeted deletion of mouse Rad1 leads to deficient cellular DNA damage responses

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.

Foil-like receptor agonists induce apoptosis in mouse 3-cell lymphoma cells by altering NF-κB activation

Toll-like receptor 9 （TLR9） recognizes microbial DNA containing unmethylated cytosyl guanosyl （CpG） sequences, induces innate immune responses, and facilitates antigen-specific adaptive immunity. Recent studies report that in addition to stimulating innate immunity, TLR9 ligands induce apoptosis of TLR9 expressing cancer cells. To understand the mechanism of TLR9-induced apoptosis, we compared the effects of CpG containing oligodeoxynucleotides （CpG ODN） on a mouse B-cell lymphoma line, CH27, with those on mouse splenic B cells. CpG ODN inhibited constitutive proliferation and induced apoptosis in the CH27 B-cell lymphoma line. In contrast, CpG ODN-treated primary B cells were stimulated to proliferate and were rescued from spontaneous apoptosis. The induction of apoptosis required the ODNs to contain the CpG motif and the expression of TLR9 in lymphoma B cells. A decrease in Bcl-xl expression and an increase in Fas and Fas ligand expression accompanied lymphoma B-cell apoptosis. Treatment with the Fas ligand-neutralizing antibody inhibited CpG ODN-induced apoptosis. CpG ODN triggered a transient NF-κB activation in the B-cell lymphoma cell line, which constitutively expresses a high level of c-Myc, while CpG ODN induced sustained increases in NF-κB activation and c-Myc expression in primary B cells. Furthermore, an NF-κB inhibitor inhibited the proliferation of the CH27 B-cell lymphoma line. Our data suggest that the differential responses of lymphoma and primary B cells to CpG ODN are the result of differences in NF-KB activation. The impaired NF-KB activation in the CpG ODN-treated B-cell lymphoma cell line alters the balance between NF-κB and c-Myc, which induces Fas/Fas ligand-dependent apoptosis.

Energetic particles’fluxes and dose in the Radiation Gene Box measured by space radiation detector onboard SJ-10 satellite

Background To evaluate the hazard of space radiation posing to the tissues,it is important to obtain exact fluxes of different radiation particles.The Radiation Gene Box(RGB)onboard SJ-10 spacecraft was an instrument designed to investigate the effects of space environment on the mESCs and drosophila.To derive the dose received by the tissues inside the RGB,the Space Radiation Detector(SRD)was installed inside it.Purpose The SRD was designed to derive the fluxes of electron,proton,hellion and gamma rays around it.If the type of the particles,the energies,the fluxes and the conversion coefficients are known,the dose received by the tissues could be evaluated.Methods The SRDwas designed as a △E-E solid-state telescope.By measuring the energy deposited in the three subdetectors,the particles’type and their energies could be discriminated.The data of SRDwere divided into 15 bins by the types of particles and their energy ranges.Results The gamma ray flux was higher than any other particle flux inside the RGB,and the electron was the most intense charge particle,while the helium ion was the most harmful radiation to the cells inside the RGB.Conclusion The dose rate inside the Radiation Gene Box was much higher than in the ground,but the integral dose of 12 days inside the RGB was about 2.13 mSv.It seemed unlikely to have obvious biological effects on the tissues of mice and drosophila.

Affinity maturation of anti-TNF-alpha scFv with somatic hypermutation in non-B cells

Activation-induced cytidine deaminase(AID)is required for the generation of antibody diversity through initiat-ing both somatic hypermutation(SHM)and class switch recombination.A few research groups have success-fully used the feature of AID for generating mutant li-braries in directed evolution of target proteins in B cells in vitro.B cells,cultured in suspension,are not con-venient 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-αscFv(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.