RESUMO
Objective: To study the characteristics of mouse embryonic stem (ES) cell line R1 expressing green fluorescent protein (GFP) efficiently and stably. Methods: Four kinds of biological characteristics: growth curve, express level of alkaline phosphatase, karyotype and pluripotentiality of the subclones of R1 ES cell expressing GFP were observed and analysed. Results: No obvious differences between R1 ES cells and the 4 ES cell clones expressing GFP［ESG（+）］ on the proliferation speed, differentiation state and the ratio of normal karyotype were observed. Teratocarcinoma concluded 3 germinal layers could form after inoculating ESG(+) cells to nude mice. Conclusion: The expression of GFP may not make any detectable effect on the proliferation speed, differentiation state and the pluripotentiality of R1 ES cells. This research work ensures efficient utilization of GFP as a reporter gene tracing ES cell in vivo.
RESUMO
Objective: To study the factors effecting the expression of the reporter green fluorescent protein (GFP) gene in the mouse embryo stem cell line R1. Methods: Three different kinds of GFP euko-expression vectors were constructed, and the expression efficiency was contrasted both at mRNA and protein levels after they were integrated into the chromosomes of host cells. Results: At protein level, the GFP expression level of the colonies transfected by the expression vector-pEF-GFP with the promoter of the peptide elongation factor (EF) were significantly higher than that of the colonies transfected by pCMV-GFP with CMV promoter and by pdCMV-GFP with double copies of CMV-GFP expression unit. There was no significant difference between the colonies transfected by pCMV-GFP and pdCMV-GFP. The detection results on mRNA level of GFP had the same tendency as that at protein level. Conclusion: (1) GFP gene expression efficiency controlled by EF promoter is distinctly higher than that by CMV promoter in NIH3T3 and R1 ES cell line.(2) A slight increase of the copy number of the foreign gene expression units in the host chromosome can not make obvious increase of its expression efficiency. (3) The vector express GFP in R1 ES cell line efficiently and stablely is obtained.
RESUMO
Objective: To study the characteristics of mouse embryonic stem (ES) cell line R1 expressing green fluorescent protein (GFP) efficiently and stably. Methods: Four kinds of biological characteristics: growth curve, express level of alkaline phosphatase, karyotype and pluripotentiality of the subclones of R1 ES cell expressing GFP were observed and analysed. Results: No obvious differences between R1 ES cells and the 4 ES cell clones expressing GFP［ESG（+）］ on the proliferation speed, differentiation state and the ratio of normal karyotype were observed. Teratocarcinoma concluded 3 germinal layers could form after inoculating ESG(+) cells to nude mice. Conclusion: The expression of GFP may not make any detectable effect on the proliferation speed, differentiation state and the pluripotentiality of R1 ES cells. This research work ensures efficient utilization of GFP as a reporter gene tracing ES cell in vivo.
RESUMO
Objective: To study the factors effecting the expression of the reporter green fluorescent protein (GFP) gene in the mouse embryo stem cell line R1. Methods: Three different kinds of GFP euko-expression vectors were constructed, and the expression efficiency was contrasted both at mRNA and protein levels after they were integrated into the chromosomes of host cells. Results: At protein level, the GFP expression level of the colonies transfected by the expression vector-pEF-GFP with the promoter of the peptide elongation factor (EF) were significantly higher than that of the colonies transfected by pCMV-GFP with CMV promoter and by pdCMV-GFP with double copies of CMV-GFP expression unit. There was no significant difference between the colonies transfected by pCMV-GFP and pdCMV-GFP. The detection results on mRNA level of GFP had the same tendency as that at protein level. Conclusion: (1) GFP gene expression efficiency controlled by EF promoter is distinctly higher than that by CMV promoter in NIH3T3 and R1 ES cell line.(2) A slight increase of the copy number of the foreign gene expression units in the host chromosome can not make obvious increase of its expression efficiency. (3) The vector express GFP in R1 ES cell line efficiently and stablely is obtained.