Expression and localization of s-lap/GFP fusion protein in Hela cell lines
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Objective To study the expression and localization of s-lap/GFP fusion protein in Hela cell lines. Methods According to the coding area sequence of s-lap gene, stop codon located at 3′terminus of s-lap gene was mutated by PCR and fused with GFP gene. Then the s-lap/GFP fusion gene was transfected into Hela cells. The expression of s-lap/GFP fusion protein was observed under fluorescence microscope. Results DNA sequencing demonstrated that s-lap/GFP recombinant vector was constructed successfully and its fusion protein was expressed in Hela cells. The observation under fluorescence microscope showed that s-lap/GFP fusion protein was spread in entire cells, but expressed highly in the nucleus. Conclusion The s-lap/GFP fusion protein can express in Hela cells and locate in entire cells, but express highly in the nucleus.Keywords:
HeLa
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Construction of green fluorescent protein retroviral vector and its expression in various cell types
Aim To achieve efficient, and stable expression of green fluorescent protein(GFP) reporter gene in tissue-engineered cells. Methods GFP retrovirus expression vector was constructed and transfected into packaging cell PT67. After G418 selection and cell cloning, cell clones producing high levels of recombinant virus were obtained, The viruses were used to infect target cells directly. Results Bright green fluorescence of the transfected cells can be observed under fluorescent microscopes 48 hrs after transfection. The transfection rates were 20%～50%. The effective expression of GFP in target cells infected by recombinant retrovirus has lasted for 2 months. Conclusion The construction of recombinant retrovials GFP vector can provide a simple, sensitive and reliable tool for labeling tissue engineered cell and cell dynamic research.
Cloning (programming)
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Objective:To construct human BNIP3 fuse protein vector,express it in Hela cells,and determine its location in the cells.Methods: The expression vector was constructed by cloning the coding sequences of fusion protein of human BNIP3 and HA onto vector pcDNA3 by two-step method;The constructed vector was then transfected into Hela cells and observed with fluorescence microscope.The recombinant plasmid was verified by enzyme digestion,polymerase chain reaction(PCR) and sequence analysis.Results: The fusion protein was highly expressed in Hela cells.Being observed with fluorescence microscopy,the fusion protein of human BNIP3 and HA was found to distribute in the mitochondria.Conclusion: The expression vector of BNIP3 fusing to HA is successfully constructed and the fusion protein has expressed in the mitochondria of Hela cells,which would facilitate the further study of BNIP3.
HeLa
Cloning (programming)
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Objective Studing the effect of erythroid-specific promoter can effectively drive expression of green/red fluorescence protein(GFP/RFP) in K562 cells.Methods Erythroid-specific promoter(β globin promoter) was amplified by PCR from human genome DNA,which was cloned into pcDNA vector to drive GFP/RFP gene expression.Then the vectors were transfected into K562 cells to prove the effect of the promoter by using fluorescence microscopy and RT-PCR.Results Fluorescence microscopy showed that GFP/RFP can strongly express in K562 cells.And the mRNA was also shown to express effectively by RT-PCR analysis.Conclusion The erythroid-specific promoter can obviously drive gene expression in K562 cells,which is valuable for gene therapy to leukemia as well as other hematology diseases.
K562 cells
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To construct novel gene AngRem104/EGFP fusion gene in eukaryotic expression vector and to investigate its expression in COS 1 cells, human AngRem104 ORF was amplified from pGEM T AngRem104 by PCR and inserted into plasmid pEGFP N1. Using lipofectin method, the recombinant expression plasmid AngRem104 pEGFP N1 was transfected into COS 1 cells. The results showed that AngRem104/EGFP expressed a fusion protein with green fluorescence protein in COS 1 cells as defined by Western blot analysis. This fusion protein was localized in the nucleus of COS 1 cells as detected by fluorescence microscopy. The use of GFP for cell marking in novel gene AngRem104 demonstrated a new technology for functional study of novel gene AngRem104.
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AIM:To construct a hAR and GFP fusion gene vector and to observe the AR-GFP gene expression in Hek293 cells. METHODS: A recombined vector pcDNA3.1/myc-HisA-AR-GFP (pH-AG) was constructed by gene engineering technique. The recombined vector was transfected into Hek293 cells using calcium phosphate. RESULTS: AR-GFP fusion protein was successfully expressed in Hek293 cells without biologic activity, which was confirmed by fluorescence microscopy and Western blotting. CONCLUSION: The Hek293 cells transfected by AR-GFP fusion gene can express its protein successfully. However, it is not a cellular model for ARI screening.
HEK 293 cells
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Objective To construct a shuttle plasmid for co-expression of intracellular pathogen resistance gene 1(Ipr1)and green fluorescent protein(GFP)gene in human lung cancer A549 cells. Methods Ipr1 and GFP genes were amplified from plasmids pEGFP-C1-Ipr1 and pEGFP-C1 by PCR respectively. The amplified Ipr1 and GFP genes as well as mycobacterium replicon OriM were cloned into eukaryotic co-expression vector pBudCE4.1, and the constructed shuttle plasmid pBud-GFP-OriM-Ipr1 was transfected to A549 cells in mediation of liposome. The expression of GFP was observed by fluorescent microscopy, and that of Ipr1 protein was determined by immunohistochemical assay. Results Both restriction analysis and sequencing proved that shuttle plasmid pBudGFP-OriM-Ipr1 was constructed correctly. Expressed GFP was observed by fluorescent microscopy in transfected A549 cells. Immunohistochemical assay proved that Ipr1 protein was expressed in transfected A549 cells and located in cell nucleus. Conclusion A shuttle plasmid for eukaryotic co-expression of Ipr1 and GFP genes was successfully constructed, which laid a foundation of further study on function of Ipr1 against tuberculosis.
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【Objective】To construct the retroviral vector carrying enhanced green fluorescent protein (EGFP) gene and to observe the expression of green fluorescent protein (GFP) in vitro and its effect on biological characteristics of target cells. 【Methods】 EGFP gene fragment, which was ligated into the multiple cloning site (MCS) with pEGFP-C1 vector preserved completely, was obtained through PCR reaction using pEGFP-C1 plasmid as a template and directionally inserted into MCS of retroviral vector pLXSN. The right recombinant retroviral vector pLXSN-EGFP was selected to transfect packaging cell PA317 to gain virus particles with infection ability. Human glioblastoma mutiforme cells SW038-C2 were transfected. The expression of EGFP gene as reporter gene in vitro and its effect on the biological characteristics of target cells were observed by fluorescence microscope and fluorescence activated cell sorting (FACS). 【Results】 The recombinant retroviral vector with EGFP gene was cloned successfully. Packaged by packaging cell PA317, the recombinant vector could transfect human glioblastoma mutiforme cells SW038-C2 effectively, express stably, and did not show inhibitory effect on the growth of target cells. Target cells with high expression of GFP became long and thin. FACS showed about 98% of these cells expressed GFP, and the intensity of the fluorescence did not decrease after culture in vitro for 6 months. The shape of target cells expressing low level GFP did not change and 30% of them expressed GFP was observed in FACS. 【Conclusions】 The transfection rate of virus vector being detected is lower than actual results while GFP is used as reporter gene. The constructed vector pLXSN-EGFP can express stably in vitro, which are useful for future research for inserting another therapy gene in the vector.
Cell Sorting
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Objective To construct the recombinant retroviral vector which carries enhanced green fluorescent protein(GFP) gene, analyze the level of GFP expressing in vitro and in vivo and observe the biological character of cell expressed EGFP gene. Methods EGFP gene was amplified by PCR and directionally inserted into the multiple cloning site of retroviral vector pLXSN. Positive clone was identified by enzyme digesting and PCR. Transfected replication defective retroviral vector was packaged by PA317 cells into SW038-C2 cells. The expression of EGFP gene in tumor cells and the biological character changing of tumor cells was observed in vitro and in vivo by fluorescence microscope and FACS. Results Successfully constructed the recombinant retroviral vector pLXSN-EGFP and was packaged by PA317. The recombinant vector can transfect the glioblastoma mutiforme cells of human SW038-C2 and express normally. The growth of transfected cells and tumor nods were not affected. The cells expressing high level EGFP gene became long and thin. Assaying by FACS, about 98% of these cells expressed GFP. The intensity of the fluorescence was not reduced after 6 months cultured in vitro. The shape of cells expressing low level EGFP gene is normal and 30% of them expressed GFP. Conclusion The EGFP gene could express normally in vitro and in vivo and didn't influence the growth of tumor cells. But the morphology of tumor cells expressed GFP gene was changed. Using GFP as the report gene, transfection rate of the retroviral vector is lower than expected.
clone (Java method)
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Objective To construct pEGFP-HPV16 E6 fusion gene in eukaryotic expression vector and to investigate its expression in Hela cells.Methods HPV16 E6 ORF was amplified from PCR2.1-HPV16E6 by PCR and inserted into plasmid pEGFP-C2.Using lipofectin method,the recombinant expression plasmid pEGFP-HPV16 E6 was transfected into Hela cells.Conclusions The results showed that pEGFP-HPV16 E6 expressed a fusion protein with green fluorescence protein in Hela cells as defined by Western blot analysis.This fusion protein was localized in the nucleus of Hela cells as detected by fluorescence microscopy.
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Objective:To construct the eukaryotic expression vector pcDNA3GFP for determination of expression of green fluorescent protein(GFP).Methods:GFP cDNA was obtained from plasmid pCIGFP with single Not I digestion and was inserted into the same restriction site of pcDNA3GFP. The upright insertion was determined with BamH I digestion.Results:With transfection of pcDNA3GFP encapsulated by Lipofect AMINE PLUS TM into the cultured MDCK cells,G418 was used for selection of positive clones expressing green fluorescence. GFP positive clones of MDCK cells formed 20 days after transfection with fluorescent microscopy.Conclusion:It was demonstrated that GFP was successfully expressed in MDCK cells,implying that GFP was a good reporter and selection marker molecule in mammalian cells.
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