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Molecular Cloning and Characterization of cDNA for Human G-CSF and Its Expression in Mammalian Cells

Author: LiFeiYu
Tutor: SunZongTang
School: Peking Union Medical College , China
Course: Molecular Immunology
Keywords: Human G-CSF Polymerase chain reaction ( PCR ) Retroviral vectors Transformation of mammalian cells Mammalian cell expression Virus packaging cell Gene mutation
CLC: R392
Type: PhD thesis
Year: 1992
Downloads: 76
Quote: 0
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Abstract


Granulocyte colony-stimulating factor(G-CSF) is one of members of the CSF family that regulate the proliferation and differentiation of hematop-oietic cells, which is produced by many kinds of human cells and some tumor cell lines. G-CSF possesses the ability to stimulate the colony formation of granulocyte from committed hematopoietic stem cells. It may exert effects on possible proliferation and differentiation of some human and murine myeloid leukemic cells.In the present study, the cDNA of human G-CSF gene was cloned following PCR amplification. Its structure was characterized by southern blotting and sequencing analysis. Its functional aspects have been studied following retroviral mediated gene transduction into SP2/0, a murine myoloma cell line.Total RNA was extracted from human bladder carcinoma cell line 5637 by Guanidine isothiocynate/CsCl method. The first-strand cDNA for G-CSF was synthesized and then amplified by using GeneAmp RNA PCR system with two specific primers synthesized in this laboratory. The amplified product, a clear band around 630 bp, was recovered following electrophoresis. The DNA product after enzyme restrictions was first cloned into Ml3mp18, then the G-CSF gene fregments in M13mp18 were claved and inserted into M13mp19 by same method, and sequencing was done in both directions following Sanger ’s dideoxy method. Sequencing date showed that the cloned gene included the intact coding region of human G-CSF cDNA which was 612 bp in length, encoding 30 amino acids of signal peptide and 174 ami no acids of mature protein of G-CSF. The sequence was identical to that reported in the literature, except one base mutation of the 43rd codon resulting in the replacement of histidine by tyrosine.The human G-CSF cDNA was isolated from M13mp19 and inserted into retroviral vector XM-6 following appropriate enzyme digestions, modification and ligation. The recomhinant retroviral vector(XM6-GCSF) was amplified in E.Coli. Restriction mapping and southern blotting demonstrated that the G-CSF cDNA was inserted into the vector in correct orientation at expected site. The recombinant vector was then introduced into amphotropic packaging cell line PA317 by lipofectin method. Individual G-418 resistant cell clones were selected and titered for virus productivity.

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