• Journal of Microbiology and Biotechnology
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• v.18 no.7
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• pp.1342-1351
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• 2008

The feasibility of the use of Flp-mediated cassette exchange in the development of a CHO cell line, which produces erythropoietin (EPO) stably and largely, was investigated. A stable, high enhanced green fluorescence protein (EGFP)-producing clone was screened by extensive flow cytometric analysis. An EPO expression unit was targeted into the premarked locus of the stable parental clone by Flp-mediated cassette exchange and a correctly targeted clone (FC28T7) was obtained. The EPO production of FC28T7 was proven to be stable in long-term culture. Furthermore, the Flp-mediated cassette exchange did not alter the stable parental clone's characteristics concerning transgene expression level and stability. Taken together, the data obtained here indicated that the establishment of CHO cell lines stably producing a desired protein is achievable using Flp-mediated cassette exchange.

• Korean Journal of Microbiology
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• v.25 no.3
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• pp.198-204
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• 1987

Biparental clones and recombinant clones were obtained by spheroplast fusion of Pseudomonas putida KU218R-3 and P.putida KU428. Formation of the fusion product was the most effective when the Pseudomonas spheroplast mixture were treated with 40% plyethyleneglycol(PEG) 6000 for 10min at room temperature, The fusants which selected by indirect method were obtained at an average frequency of 10.8%. Most of the fusants were biparental clones (10.4%) and the recombinant clones were produced in low yield (0.42%). Fusants, at the frequency of 4% were obtained without PEG 6000, which shows that fusion is not strictly dependant on PEG. The stability of fusants were examined. Most of the biparental clones were segregated to parental form amd late recombinants were formed on further propagation of biparental clone but the recombinant clones were nery stable.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.9
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• pp.4827-4834
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• 2012

Tumor suppressor genes have received much attention for their roles in the development of human malignancies. Gelsolin has been found to be down-regulated in several types of human cancers, including leukemias. It is, however, expressed in macrophages, which are the final differentiation derivatives for the monocytic myeloid lineage, implicating this protein in the differentiation process of such cells. In order to investigate the role of gelsolin in leukaemic cell differentiation, stable clones over-expressing ectopic gelsolin, and a control clone were established from U937 leukaemia cells. Unlike the control cells, both gelsolin-overexpressing clones displayed retarded growth, improved monocytic morphology, increased NADPH and NSE activities, and enhanced surface expression of the ${\beta}$-integrin receptor, CD11b, when compared with the parental U937 cells. Interestingly, RT-PCR and western blot analysis also revealed that gelsolin enhanced p21CIP1 mRNA and protein expression in the overexpressing clones. Moreover, transient transfection with siRNA silencing P21CIP1, but not the control siRNA, resulted in a reduction in monocytic differentiation, accompanied by an increase in proliferation. In conclusion, our work demonstrates that gelsolin, by itself, is capable of inducing monocytic differentiation in U937 leukaemia cells, most probably through p21CIP1 activation.