[Transfection of the nm23-H1 gene into BcaCD885 cell line inhibits the potential of invasion, adhesion and mobility].
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To transfect nm23-H1 into the BcaCD885 cell lines in order to get safe high-efficiency and low-toxicity, and to find out whether nm23-H1 could affect the invasion and metastases ability of BcaCD885 cell lines.Lipofect was used to transfect nm23-H1 into BcaCD885 cell lines; immunohistochemistry was used to detect the difference expression of nm23-H1 between transfected and non-transfected cell lines; then transwell-room and wash way were used to detect the difference of invasion and metastases ability between transfected and non-transfected cell lines.PCMV-NEO-BAM system gave the stability expression of nm23-H1; there was significant different NDPKA expression between transfected and non-transfected BcaCD885 cell lines; the invasion and metastases ability of transfected BcaCD885 cell lines decreased obviously.nm23-H1 can inhibit the metastases of BcaCD885 cell lines significantly.Cite
Objective To investigate the amelogenin expression of recombinant plasmid PcDNA3-AMG in COS-1 cell line.Methods PcDNA3-AMG was used to transfect COS-1 cells firstly.An appropriate concentration of Zeoin was chosen for the selection of stably transfected cell colonies.The ELISA technique was applied to detect the expression of recombinant amelogenin.Results The amelogenin was detected in the stably transfected COS-1 cells in both cell culture supernatant and cell homogenate,and the concentration of expressed amelogenin in PcDNA3-AMG transfected cell homogenate was as high as 0.253 μg/ml.Conclusions The results provided basis for the preparation of recombinant AMG by PcDNA3-AMG transfected eukaryrotic cell line COS-1.
Amelogenin
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Objective To establish a PC-3 cell line stably and highly expressing BRP 44. Methods mRNA sequence of BRP 44 was downloaded. A pair of primers containing the sites of given restrictive endonuclease at both the 5 ends were designed by Oligo 6 and synthesized. Reverse transcription PCR of the total RNA extracted from LNCaP cell line was performed. The products of PCR were cloned into a highly efficient eukaryotic expression vector pcDNA 3. 1/ myc-His A. The recombinants were sequenced and identified by restrictive endonuclease digestion and then transfected into the PC-3 cell line by lipofectamine 2000. The stable transfectants were screened by G 418. Cell subclones were isolated by gradient dilution. The cell subclones highly expressing BRP 44 were identified by northern blotting. Results The eukaryotic expression vector stably and efficiently expressing BRP 44 were constructed and the cell subclones stably and efficiently expressing BRP 44 were established. Conclusion The cell subclones stably and efficiently expressing BRP 44 can be used for the further study of the effect of BRP 44 on prostate cancer cell biological behavior.
Lipofectamine
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Objective:To study the effect of liposome targeting ribonuclease inhibitor(RI) on the proliferation and cell cycle of breast cancer cell line MDA-MB-231.Methods:By the induction of liposome,pLNCX-RI and eukarytic expression vectors pLNCX were transfected into breast cell line MDA-MB-231.Then the subclone cell lines MDA-MB-231-231/pLNCX-RI which high expressed RI stably were obtained by persistent G418 selection.Western-blot and RT-PCR were used to detect RI expression level in transfected cell line;Cell prolifetation was analyzed by MTT assay;Cell cycle was analyzed by flow cytometry.Results:The subclone cell line MDA-MB-231-231/pLNCX-RI which high expressed RI stably was successfully selected.MTT showed the cell proliferation was inhibited(P0.01).Cells stopped at G0/G1 phase and cell number in S phase was sharply decreased by the induction of liposome(P0.01).Conclusions:The malignant proliferation of breast cancer cells is inhibited by RI.
MTT assay
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The objective of the present study is to establish the Marc-145 cell line that stably expresses porcine poFcγRⅢ(porcine Fc gamma receptor Ⅲ,poFcγRⅢ).The total RNA was extracted from porcine alveolar macrophage(PAM),and poFcγRⅢ and γ-chain cDNAs were cloned by RT-PCR,then they were inserted into the eukaryotic expression vectors pcDNA3.1(+)and PIREShyg3 respectively.The marc-145 cell line was stably transfected with pcDNA3.1-poFcγRⅢ and PIREShyg3-γ plasmids by Lipofectamine2000,then the co-transfected cells were selected by HygromycinB(300 mg/L)and G418(400 mg/L).The expression of poFcγRⅢ on transfected cells was verified through RT-PCR,rosetting test and FCM.The eukaryotic expression vectors were successfully constructed and the Marc-145 cell line with stable poFcγRⅢ expression was obtained.The Marc-145 cell transfected with the poFcγRⅢ cDNA were able to bind porcine IgG.
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Immunofluorescence
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Objective To search the appropriate experimental conditions for using green fluorescent protein (GFP) as a reporter gene in tumor gene therapy. Methods The plasmids carrying mutated GFP gene were transfected into the eukaryotic cells to observe transient gene expression. These studies were conducted to 1. directly determine GFP expression and express stability in the COS-7 cells, 2. compare the transfection efficiency of two plasmids pcDNA 3-EGFP, pSVK 3-S65T with different promoters in different tumor cell lines, 3. determine two genes expression in a single cell using LacZ cotransfected with GFP by FACS. Results Fluorescence could be detected in intact viable cells under different sets of conditions. The expression of GFP might last two weeks or more and the expressed fluorescence was stable. The transfection rate of pcDNA 3-EGFP expressed was different in three tumor cell lines examined. But pSVK 3-GFP expressed similarly in four tumor cell lines examined. FACS showed the probability of two genes entering a single cell is above 85% at the ratio 1∶4. Conclusion The above data indicate that the GFP can be visualized continuously and directly for gene expression in living cells. GFP may also be used for quicklly selecting cells carrying a target gene.
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AIM: to establish the cell lines whose hREV3 gene expression was blocked by antisense RNA and observe their characteristis of cell growth rate and N - methyl - N' - nitrosoguanidine (MNNG) sensitivi- ty . METHODS: With modified calcium phosphate - DNA coprecipitation method the eukaryocytic expression plasmid expressing antisense fragment of hREV3,pBK - RSV - hREV3- and pMAMneo - amp hREV3 were transfected into human embryo kidney cell line of HEK - 293. After G418 selection, cell lines of 293 - B - hREV3- and 293 - M hREV3- were established. By cell counting method, the cell growth rate and MNNG sensitivity of these cell lines were characterized. RESULTS: No change of cell growth rates of these cell lines was observed whether hREV3 gene expres- sion was blocked by either the persistent or induced expression of the antisense hREV3 RNA. While the sensitivity of these cell lines to MNNG was somewhat elevated, as compared with their parent cell line 293 and the cell lines trans- fected with vector DNAs. CONCLUSION: The gene product of hREV3 was not essential for the cell growth, but it may play a role in the DNA repair functions of the cells after exposure to DNA damaging agents.
HEK 293 cells
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Objective To investigate the apoptosis in human lung carcinoma cell line A549 induced by recombinant Caspase 3 gene.Methods The expression vector pEGFP C1 with caspase 3 gene was constructed and transferred into the A549 cell line.The expression level of caspase 3 gene in A549 cell line was detected by RT PCR method and immunohistochemical staining.The cell morphological changes were observed under fluorescent microscopy and electron microscopy.The cell survival rate was assayed by MTT method.Results The expression of caspase 3 gene in A549 cells was detectable by RT PCR method and immunohistochemical staining 48 h after transfection.The cell morphological observation and MTT showed the apoptosis in A549 cell line and the cell growth curve declined 24 48 h after transfection.Conclusion Recombinant Caspase 3 gene can effectively induce A549 cells to apoptosis.
MTT assay
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It is assumed that a cell that is transfected for any gene addition or replacement or was premarked with a cell tracker dye retains the characteristics of the original cell. The following experiments compare the original C6 rat glioma cell line with C6 cells transfected with the retroviral plasmid LacZ, and the human glioma cell lines GaMG, U373, U251, and D54 with cells stained with tracker dyes (Dil and DiO). We tested adhesion, migration and proliferation. C6 cell transfection did not affect adhesion but decreased (p < 0.05) migration. Dil staining resulted in a significant decrease (p < 0.01) in adhesion in all cell lines but U251. After DiO staining human cell lines U373 and D54 displayed a decrease in adhesion (p < 0.01) whereas U251 and GaMG cells had enhanced adhesion (p < 0.01). Dye marking of C6, GaMG and U373 cells did not alter migratory capacity. In contrast, Dil and DiO reduced migration of U251 and D54 cells (p < 0.05). There was a decrease (p < 0.01) in proliferation of the human cell lines after Dil staining. Transfection or membrane dyes can alter basic cell characteristics. The assumption that a transfected or dye marked cell is the same as the original cell but with an additional gene or the presence of a dye in the membrane is untenable.
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[Objective] To transfect nm23-H1 gene into the BcaCD885 cell lines , and observe whether nm23-H1 can affect the invasion and metastatic behavior of BcaCD885 cell lines. [Methods] nm23-H1 gene was transfected into BcaCD885 cell lines using lipofect technique. The difference in expression of nm23-H1 between transfected and non-transfected cell lines was dectected by immunohisto-chemistry. The difference of invision and metastatic behavior was detected by transwell-room and wash way methods. [Results] Stable expression of nm23-H1 was achived by using PCMV-NEO-BAM system. Significant difference in NDPKA expression between transfected and non-transfected of BcaCD885 cell lines was founded; The cell control number of transfected and non-transfected cells which showed the ability of invasion was 41. 1% ± 1. 2% and 64. 5%±6. 3% respectively, while the chemical-trend moving ability was 50. 0% ± 6. 3% and81. 0% ± 3. 9%. The adhesion ability of transfected BcaCD885 cell lines decreased significantly. [Conclusion] nm23-H1 can significant inhibit the metastases of BcaCD885 cell lines.
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