References

Notes: Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan 0006-291X ENGLISH UNITED-STATES Two steps of gene targeting were used to replace the p53 gene with the E. coli beta-galactosidase (lacZ) gene in mouse embryonic stem (ES) cells. The first targeting vector consisted of neo and herpes simplex virus thymidine kinase (HSV-tk) genes as a neo-tk cassette in the middle of the targeting vector. At the first targeting, the homologous recombinants became G418 resistant and ganciclovir (GANC) sensitive and were selected by G418 alone. At the second targeting, homologous recombination reciprocally exchanged the neo-tk casette in the ES cell chromosome with the lacZ fragment in the second targeting vector and thus made the ES cells GANC resistant. We obtained two ES cell clones, in which the p53 gene for both had been replaced with a totally non-homologous sequence of the lacZ gene. The germ- line transmission of the manipulated ES cells also demonstrated that the entire procedure had no detrimental effects on ES cells at all l; a; c; Z; neo JOURNAL-ARTICLE EC 3.1.21.-; EC 3.2.1.23; 0; 0 94324974 9411
Reference ID: 4979

Notes: [Abstruct] Here we report that we could obtain a highly differentiated smooth muscle cell line by screening the expression of alpha-smooth muscle actin from p53 knock out mice aorta. This cell revealed extended bipolar shape and expressed h-caldesmon and calponin as well as a-smooth muscle actin as protein markers of differentiated smooth muscle. Further intracellular calcium increase was induced by application of noradrenaline in a dose dependent manner and calcium oscillation was also observed in a higher dose (100uM). Appropriate appllication of 5-azacytidine enhanced these tendencies and induced slow contraction by endothelin-1 and phenylephrine. [Establishement of new smooth muscle cell line] The thoracic aorta of three monts old p53 dificient (-/-) mice was dissected and connetive tissue as well as fat were removed. After endothelial layer was rubbed out, smooth muscle layer was cut into small pieces and incubated in the medium containing 20U/ml elastase (Sigma) and 1mg/ml collagenase (Sigma) at 37 C for 2 hours. The tissue suspension was filtered with a nylon filter, centifuged at low speed for short time, and the pellet was resuspended in growth medium (DMEM supplemented with 10% fetal bovine serum, 100U/ml penicillin, and 0.1 mg/ml streptomycin). The cells were incubated in humidified 5% CO2-95% air atmosphere at 37 C. The cell was cloned by plating 10 cells per one well in a 96 well plate and incubated in the growth medium. Then each colony was transferred to a 24 well plate and cultured. Individual cells were further subcultured and the cells expressing a-smooth muscle actin were selected by immunoblotting with the antibody. These cells were subcultured, multiplied, and cloned again. Finally the cells expressing h-caldesmon induced by 5-azacytidine were selected.
Reference ID: 4978