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2009•Journal of University of South ChinaRequires access

Construction and Identification of M-CSF Stably Expressing Cell Line in Cytoplasm

Zhihong Yao

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Abstract

Objective To establish a cell line that stably expresses cytoplasmic M-CSF to explore the further effect of cytoplasmic M-CSF. Methods The constructed pCMV/cyto/myc-M-CSF plasmid was identified by PCR and double digestion.Both pCMV/cyto/myc and pCMV/cyto/myc-M-CSF vectors were trasfected into HeLa cells by using liposome and screening with G418.Then RT-PCR and immunocytochemistry were confirmed to detect the expression and localization of M-CSF in HeLa cells. Results The size of the recombinant vector pCMV/cyto/myc-M-CSF was 1400bp.After transfecting,high levels of both M-CSF mRNA and M-CSF protein were expressed and localized to the cytosol in M-CSF-transfected HeLa cells(P0.05),which suggested that a cell line that highly expressed cytoplasmic M-CSF was successfully established. Conclusion A cell line that highly expressed cytoplasmic M-CSF was established.

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What this paper is about

Objective To establish a cell line that stably expresses cytoplasmic M-CSF to explore the further effect of cytoplasmic M-CSF. Methods The constructed pCMV/cyto/myc-M-CSF plasmid was identified by PCR and double digestion.Both pCMV/cyto/myc and pCMV/cyto/myc-M-CSF vectors were trasfected into HeLa cells by using liposome and screening with G418.Then RT-PCR and immunocytochemistry were confirmed to detect the expression and localization of M-CSF in HeLa cells. Results The size of the recombinant vector pCMV/cyto/myc-M-CSF was 1400bp.After transfecting,high levels of both M-CSF mRNA and M-CSF protein were expressed and localized to the cytosol in M-CSF-transfected HeLa cells(P0.05),which suggested that a cell line that highly expressed cytoplasmic M-CSF was successfully established. Conclusion A cell line that highly expressed cytoplasmic M-CSF was established.

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Available abstract

Objective To establish a cell line that stably expresses cytoplasmic M-CSF to explore the further effect of cytoplasmic M-CSF. Methods The constructed pCMV/cyto/myc-M-CSF plasmid was identified by PCR and double digestion.Both pCMV/cyto/myc and pCMV/cyto/myc-M-CSF vectors were trasfected into HeLa cells by using liposome and screening with G418.Then RT-PCR and immunocytochemistry were confirmed to detect the expression and localization of M-CSF in HeLa cells. Results The size of the recombinant vector pCMV/cyto/myc-M-CSF was 1400bp.After transfecting,high levels of both M-CSF mRNA and M-CSF protein were expressed and localized to the cytosol in M-CSF-transfected HeLa cells(P0.05),which suggested that a cell line that highly expressed cytoplasmic M-CSF was successfully established. Conclusion A cell line that highly expressed cytoplasmic M-CSF was established.

Key concepts: Cytoplasm, HeLa, Transfection, Immunocytochemistry, Molecular biology, Cell culture, Recombinant DNA, Cell

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