Transition of WT1 isoforms inhibits proliferation and promotes apoptosis of human leukemia cell line HL-60

AIM: To study the potential effects of exogenous Wilms tumor 1 (WT1) isoforms on the proliferation and apoptosis of human leukemia cell line HL-60. METHODS: WT1 (17AA－/KTS－) gene obtained by RT-PCR was cloned into a PCDH1-MCS1-EF1-copGFP plasmid. The recombinant plasmid was confirmed by enzyme digestion and sequencing, and was transfected into HL-60 cells by LipofectamineTM 2000. The stable transformants were selected by G418 screening. WT1(17AA－/KTS－) expression was identified by real-time fluorescence quantitative RT-PCR and Western blotting. The proliferation of the cells was measured by MTT assay. The apoptosis of the cells was determined by morphological observation and flow cytometry analysis. RESULTS: The eukaryotic expression vector PCDH1-MCS1-EF1-copGFP-WT1 (17AA－/KTS－) was successfully constructed. The recombinant cells exhibited high mRNA and protein levels of WT1(17AA－/KTS－). The growth of recombinant cells was slower than that of HL-60 cells transfected with control vector and normal HL-60 cells. After exposed to As2O3 at 2 μmol/L for 48 h, both recombinant cells and control cells exhibited the morphological characteristics of apoptosis, but the former was more typical than the latter. The apoptosis was enhanced in the recombinant cells after the cells were exposed to As2O3 for 24 h. CONCLUSION: Exogenous WT1(17AA-/KTS-) isoform inhibits the proliferation and promotes the apoptosis of leukemic cells.     

High expression level of TAL1 gene in newly diagnosed acute myeloid leukemia

AIM: To investigate the characteristic of T-cell acute lymphocytic leukemia 1 (TAL1) gene expression in acute myeloid leukemia (AML) cell lines and in primary AML cells from de novo AML patients with different subtypes. METHODS: Real-time PCR was used to determine the expression of TAL1 mRNA in acute leukemia cell lines (Jurkat, CCRF-CEM, HL-60 and NB4 cell lines) and peripheral blood mononuclear cells from 47 newly diagnosed AML patients. Twelve healthy individuals were served as healthy control group. RESULTS: A significantly increased level in TAL1 mRNA was found in AML cell lines (HL-60 and NB4), T-cell acute lymphacytic leukemia (T-ALL) cell lines (Jurkat, CCRF-CEM) and primary AML cells compared with the healthy controls. Over-expression of TAL1 was found in all detected AML subtypes, the highest level of TAL-1 mRNA was found in AML-M1 and AML-M5 subtype (P<0.05). CONCLUSION: High expression of TAL1 in AML might influence the differentiation and proliferation of myeloid cells, further investigation needs to confirm whether it would be as a biomarker for pathogenesis of AML.     

Effect of PRL-2 gene transfection on proliferation and cell cycle in hepatocellular cell line

AIM: To observe the changes of proliferation and cell cycle after PRL-2 gene effectively expressed in human hepatocellular cell line.METHODS: The PRL-2 vector was transfected into CL1 cell with lipofectamine reagent,the stable expression clones were screened by G418.The expression of PRL-2 mRNA was detected by real-time PCR.The expressive protein was identified by Western blotting.The subcellular localization was demonstrated by immunochemistry.The cell cycle was measured by flow cytometry.The population doubling time (TD) was analyzed by MTT assay.The expressions of cyclin A,cyclin D1,cyclin E,p16,p21Waf1 and p27Kip1 were detected by Western blotting.The p21Waf1 mRNA was determined by real- time PCR.RESULTS: The full length ORF of PRL-2 gene was inserted into the vector pcDNA3.1 (+),transfected into CL1 cells,and expressed successfully.Real-time PCR showed stable expression of PRL-2 mRNA.Western blotting confirmed the overexpression of PRL-2 protein.The subcellular localization of PRL-2 was in the plasmid.The proportion of cells in S-phase was increased.The population doubling time was reduced (P<0.01),a significant decrease was observed both in the mRNA and the protein expression of the p21Waf1 in comparison with untransfected or vector- transfected control cells (P<0.05).The expressions of cyclin D1,cyclin E,cyclinA,p16 and p27Kip1 were not appreciably different between the control and PRL-transfected cell lines.CONCLUSION: Eukaryocytic expression vector of PRL-2 has been successfully constructed,which shows stable and effective expression in CL1 cell line.PRL -2 increases cell proliferation by stimulating progression from G1 into S phase,which is primarily associated with decreased p21Waf1.     

The antisense block of CROC-1 gene expression makes cells grow slower

AIM:To establish FL-CROC- 1 ^- cell line in which CROC- 1 gene expression was blocked and study the role of CROC- 1 gene in cell growth. METHODS:The appropriate length cDNA fragment of the recently identified human gene CROC- 1 which encodes ubiquitin-conjugating enzyme like protein(Ubc-like protein) was cloned into the reconstructed eukaryotic expression vector pMAMneo-amp^- by antisense strategy. The recombinant plasmid which can express CROC- 1 antisense RNA was selected by restriction enzyme map analysis. The antisense expression recombinant plasmid pMAM-antiCROC- 1 was then transfected into human amnion FL cells by a modified calcium phosphate-mediated transfection procedure and selected with MEM medium containing 400 μg/mL geneticin. Finally ,the growth rate of the G418 resistant FL-CROC- 1 ^- cell line was determined.RESULTS:When the antisense inhibition of CROC-1 gene expression was induced by dexamethasone,the growth rate of the FL-CROC-1^- cel line was obviously slower as compared with that of the control FL cell and FL-MAMneo cell which was established by transfection of plasmid pMAMneo-amp^-(P<0.05).CONCLUSIONS:FL-CROC- 1 ^- cell line was successfully established in this study and the result of FL-CROC- 1 ^- cell growth suppression suggests that CROC- 1 gene encoded Ubc-like protein plays a positive regulation role in cell growth.     

Proapoptotic mechanism and changes of endogenous TGF-β1 in NB4 cells induced by exogenous TGF-β1

AIM: To study the effects of transforming growth factor-β1 (TGF-β1) on cell apoptosis, cell cycle, production of endogenous TGF-β1, expressions of P27Kip1, cyclin E and bcl-2 mRNA levels in NB4 cells. METHODS: Apoptotic morphological changes were observed by Wright-Giemsa staining. Cell cycle and apoptosis were detected with flow cytometry. Semiquantitative RT-PCR was used to examine the mRNA levels of endogenous TGF-β1, P27Kip1, cyclin E and bcl-2. RESULTS: TGF-β1 significantly restrained the growth and promoted the apoptosis of NB4 cells. The blockage of NB4 cells treated by TGF-β1 at concentration of 5 μg/L was in G1 phase. Endogenous TGF-β1 mRNA expression in NB4 cells was up-regulated when the concentration of exogenous TGF-β1 was <5 μg/L. Meanwhile, the expression of endogenous TGF-β1 mRNA was down-regulated when the concentration of exogenous TGF-β1 was 10 μg/L. After treated with TGF-β1 at concentration of 5 μg/L, P27Kip1 mRNA expression in NB4 cells was up-regulated, cyclin E and bcl-2 were reduced. CONCLUSION: TGF-β1 is able to induce apoptosis and cell cycle distribution abnormally in NB4 cells by (1) Up-regulation of endogenous TGF-β1, so that NB4 cells was induced into apoptosis through consequently high expression of P27Kip1. (2) TGF-β1 may lead to cell cycle arrest by inhibiting the expression of cyclin E directly, or by inhibiting the activity of cyclin E through the increased expression of P27Kip1. (3) Down-regulation of bcl-2 induces apoptosis of NB4 cells.     

Fibrinolytic activity and its mechanisms in various leukemic cell lines

AIM:To study the relation between expression of uPAR and annexinⅡ and fibrinolytic activity in various leukemic cell lines. METHODS:The plasma activity was measured under the reaction between cells of NB4, SHI-1, K562, Jurkat, Raji and plaminogen by chromogenic assay. The protein expressions of uPAR and annexinⅡin cells of NB4, SHI-1, K562, Jurkat, Raji were detected by flow cytometry method. The mRNA expressions of uPAR and annexinⅡin cells of NB4, SHI-1, K562, Jurkat, Raji were detected by RT-PCR. RESULTS:The plasma activity in SHI-1 cells and NB4 cells were higher obviously than that in Raji, K562 and Jurkat cells. The protein expression ratio of uPAR and annexinⅡ in NB4 cells were (13.15±1.61)% and (95.97±1.19)%， respectively, they were (99.00±0.26)%, (90.35±2.15)% respectively in SHI-1 cells, and they were lower in K562, Jurkat, Raji cells. The expression of annexinⅡ mRNA in NB4 cells was higher than that in SHI-1 cells, and they were undectectable in K562 and Jurkat cells. The expression of uPAR mRNA in NB4 and SHI-1 cells were higher than that in Jurkat and K562 cells. The expression of uPAR mRNA in Raji cells was undectectable. CONCLUSION:The primary hyperfibrinolysis in leucocythemia cells was observed, and relation was closely with the expression of annexinⅡ. It might be the main reason for bleeding and disseminated intravascular coagulation in patients with acute promyelocytic leukemia and acute monocytic leukemia.     

Effect of WT1 silencing by siRNA on expression of Wnt/β-catenin and nephrin in mouse podocytes

AIM: To study the effect of WT1 silencing by small interfering RNA (siRNA) on podocyte vitality and expression of Wnt/β-catenin and nephrin in mouse podocytes. METHODS: Conditionally immortalized mouse podocytes were cultured at 33 ℃ in RPMI-1640 medium for proliferation and induced for differentiation at 37 ℃. The podocytes were transfected with WT1 siRNA. The cell vitality was detected by MTT assay. The expression of WT1,Wnt1,β-catenin and nephrin at mRNA and protein levels was determined by real-time qRT-PCR and Western blotting. RESULTS: WT1 siRNA induced the increase in the expression of Wnt1 at mRNA and protein levels, inhibited the phosphorylation of β-catenin, and reduced the cell vitality. Meanwhile, the expression of nephrin at mRNA and protein levels was decreased. CONCLUSION: WT1 siRNA reduces the expression of nephrin in podocytes and the vitality of the cells by activating Wnt/β-catenin signaling pathway.     

Effect of antisense VEGF gene on VEGF expression in human renal cell carcinoma

AIM: To construct eukaryotic expression vector carrying human antisense VEGF gene and to study its effect on VEGF expression and growth of renal cell carcinoma. METHODS: VEGF gene was cloned. Antisense VEGF gene was inserted into eukaryotic expression vector pcDNA3.1(-), then using restrict enzyme to confirm the result. The vector was transfected into renal cell carcinoma and positive clone was selected by using G418. The VEGF expression was detected by using RT-PCR and immunohistochemical method. The growth of cells was measured by MTT and cell cycle by FCM. RESULTS: Antisense VEGF gene eukaryotic expression vector was successfully constructed. Compared with empty vector group and control group, the amount of VEGF mRNA expression and VEGF protein were decreased in the antisense VEGF group, the difference was significant. There was no significant difference between empty vector group and control group. The cell growth in the antisense VEGF group became slower, and the percent of G1 phase increased and the percent of S phase decreased. CONCLUSION: Antisense VEGF gene decreases the VEGF mRNA and protein expression in renal cell carcinoma and inhibits the growth of renal carcinoma. Antisense VEGF gene may be used in gene therapy of renal carcinoma.     

Effects of c-myb antisense RNA on the proliferation and collagen Ⅰ gene expression in cultured hepatic stellate cells

AIM:To investigate the effects of c-myb antisense RNA on the proriferation and collagen Ⅰ gene expression in cultured hepatic stellate cells(HSC) in rats.METHODS:The c-myb antisense gene recombinant retroviral vector(pDOR-myb) was constructed, and then was transfected into retroviral package cell line PA 317 by means of N-[1-(2,3-Dioleoyloxy) propyl]-N, N, N-trimethylammonium methyl-sulfate(DOTAP) liposomal transfection reagent. The pseudoviruses produced from the resistant PA317 cells selected with G418 were collected, with which HSCs isolated from rat liver were infected. The cell proliferation was measured by MTT method, c-myb, α₁-Ⅰ collagen mRNA expression and c-myb protein in HSCs were detected with semi-quantitative RT-PCR and western-blot, respectively.RESULTS:HSCs from rats were isolated successfully with the viability >98%. In the pDOR-myb infected HSCs, c-myb expression levels, the cell proliferation, and α₁-Ⅰ collagen mRNA expression were repressed significantly.CONCLUSIONS:c-myb plays a key role in the activation and proliferation of HSC. c-myb antisense RNA can inhibit cell proliferation and α₁-Ⅰ collagen mRNA expression in the infected HSC. These data suggest that inhibition of c-myb gene expression would be a potential way for the treatment of liver fibrosis.     

Regulation of ghrelin on expression of ATP-binding cassette transporter A1/G1 in THP-1 derived foam cells

AIM: To investigate the regulation of ghrelin on the expression of ATP-binding cassette transporter A1 and G1 (ABCA1/ABCG1)during the foam cell formation. METHODS: The human monocytic leukemia cell line (THP-1)was chosen in our study. The differentiation of THP-1 cells into macrophages was induced by using phorbol myristate acetate (PMA). Macrophages were then incubated with oxidized LDL (ox-LDL)to generate foam cells. Ghrelin of different concentrations were treated at different time points during foam cell formation. The ABCA1/ABCG1 protein and mRNA levels were detected by Western blotting and RT-PCR. The effect of variance of cholesterol content was measured by zymochemistry via-fluorospectrophotometer. RESULTS: Ghrelin reduced the content of lipid droplet in foam cells, and increased the efflux of intracellular cholesterol significantly. Ghrelin increased ABCA1 protein mass and mRNA level in dose-dependent manner. The changes of ABCG1 protein and mRNA level were the same as ABCA1. CONCLUSION: Ghrelin interfere atherosclerosis by up-regulating the expression of ABCA1 and ABCG1.     

Effect of Bmi-1 gene overexpression on proliferation of human gastric mucosal epithelial cell line GES-1

AIM: To investigate the effect of B lymphoma Moloney murine leukemia virus insertion region 1 ( Bmi-1 ) gene overexpression on the proliferation of a human normal gastric epithelial cell line GES-1. METHODS: The plasmid containing Bmi-1 gene or empty plasmid was transfected into GES-1 cells by retroviral mediation. The expression of Bmi-1 at mRNA and protein levels was detected by quantitative real-time PCR (qRT-PCR) analysis and Western blotting, respectively. The effect of Bmi-1 gene overexpression on the cell cycle of GES-1 cells was evaluated by flow cytometry. The proliferation of the stably transfected cells was measured by Cell Counting Kit-8. RESULTS: The results of qRT-PCR analysis and Western blotting demonstrated that stably transfected cell line was successfully established. The results of flow cytometry analysis showed that overexpression of Bmi-1 reduced the G₀/G₁ phase, arrested the cells in G₂/M phase and S phase. The growth curve showed that overexpression of Bmi-1 resulted in increased growth speed. CONCLUSION: Increase in Bmi-1 gene expression regulates the cell cycle and promotes the proliferation of GES-1 cells.     

Promoter methylation status and demethylation-induced expression of SFRP genes in human acute leukemia cell lines

AIM: To investigate the relationship between promoter hypermethylation of secreted frizzled-related protein (SFRP) genes and acute leukemia (AL),and to study the mechanism how 5-aza-2-deoxycytidine (5-Aza-CdR) reverses the hypermethylation of SFRP genes in human AL cell lines. METHODS：Methylation-specific PCR (MSP) was used to detect the methylation levels of SFRP1, SFRP2, SFRP4 and SFRP5 genes in different human AL cell lines (Molt-4, Jurkat, HL-60 and NB4). The methylation levels of these genes in Jurkat cell line before and after 5-Aza-CdR treatment were also analyzed by MSP. The expression of SFRP1, SFRP2, SFRP4 and SFRP5 mRNA was detected by real-time fluorescence quantitative RT-PCR. The mRNA levels of DNA methyltransferase (DNMT) 1, DNMT3A and DNMT3B were analyzed by semiquantitative RT-PCR. RESULTS：None of normal human blood or bone marrow mononuclear cells showed methylation of SFRP genes. Hypermethylation in the promoter regions of SFRP1, SFRP2 and SFRP5 genes was observed in all of the four AL cell lines. SFRP4 gene was totally methylated in NB4, Molt-4 and Jurkat cell lines but partially methylated in HL60 cell line. Treatment with 5-Aza-CdR for 72 h successfully reversed the hypermethylation of SFRP genes, and significantly increased the mRNA expression of SFRP. Moreover, the mRNA expression of DNMT1, DNMT3A and DNMT3B was down-regulated by 5-Aza-CdR in a concentration-dependent manner. CONCLUSION：Methylated SFRP genes may serve as potential independent biomarkers for early detection of AL. 5-Aza-CdR activates SFRP gene expression by demethylation of SFRP genes and down-regulation of DNMT1, DNMT3A and DNMT3B mRNA expression.     

Effect of P2X7R gene silencing by RNA interference on proliferation and phagocytosis of murine macrophage cell line RAW264.7

AIM: To establish a cell line of stable silencing of P2X₇ receptor (P2X₇R) expression through short hairpin RNA (shRNA)-mediated interference in murine RAW264.7 macrophages, and to investigate the proliferation and apoptosis in the cell line. METHODS: Stable silencing of P2X₇R gene in the RAW264.7 cells was achieved by recombinant shRNA plasmid targeting murine P2X₇R gene via liposome mediated transfection, followed by G418 selection. The efficacy of plasmid transfection and P2X₇R silencing in G418 resistant cells was verified by immunofluorescent microscopy and real-time PCR, respectively. The proliferative activity was analyzed by CCK-8 assay and EdU cell proliferation assay. The cell cycle distribution and apoptosis were evaluated by flow cytometry. RESULTS: The expression of P2X₇R at mRNA and protein levels was down-regulated by 80% in sh P2X₇R group compared with negative control (NC) plasmid transfection. In addition, P2X₇R-silencing cells exhibited higher proliferative activity compared with NC and wild-type RAW264.7 cells (P<0.05). Compared with NC cells, P2X₇R silencing resulted in an increase in the phagocytosis of the cells (P<0.05). CONCLUSION: A cell line RAW264.7 of stable silencing of P2X₇R expression was successfully established. P2X₇R gene silencing stimulates the proliferation, and changes phagocytic function in murine RAW264.7 macrophages.     

Inhibition of proliferation in B lymphoma cell line Raji transfected with exogenous HCAP1 gene

AIM: HCAP1 gene has been mapped at human chromosome band 17p13.3, a region with high frequency of loss of heterzygosity in various types of tumor. The aim of this study was to investigate the effects of exogenous HCAP1 gene products on proliferation of B lymphoma cell line Raji. METHODS: HCAP1 gene was transfected into Raji cells. The cells stably expressed exogenous HCAP1 gene were screened with G418. The effects of HCAP1 protein overexpression on proliferation in Raji cells were assessed by viable cell count, cell growth curve, colony formation assay in soft agar, and 5-bromo-2'-deoxy-uridine (BrdU) labeling and detection assay. RESULTS: The Raji cells overexpressed HCAP1, displayed significantly slow growth rate, extended cell doubling time, decreased capacity of colony formation and reduced percentage of BrdU incorporation in the cells (P<0.05), as compared with control Raji cells which transfected with empty vector, or compared with parent Raji cells. CONCLUSION: Overexpression of exogenous HCAP1 in Raji cells inhibits the cell proliferation.     

Effects of BMI-1 over-expression on HOX family expression and cell cycle in HeLa cells

AIM: To observe whether transfection of mammalian expression vector pEGFP containing the gene of B-cell specific moloney leukemia virus insertion site 1(BMI-1) could express in human cervix cancer cell line HeLa, and to detect its effect on HOX family expression and cell cycle.METHODS: pEGFP-BMI-1 was transfected into HeLa cells with Lipofectamine 2000. The expression of pEGFP-BMI-1 was determined by EGFP fluorescence and Western blotting. SYBR green I real-time RT-PCR was used to quantitate mRNA expression of P16INK4a, hTERT, HOXA9, HOXB4 and HOXC13. FACS analysis was used to detect the change of cell cycle.RESULTS: In HeLa cells transfected with pEGFP-BMI-1, the results of real-time RT-PCR showed that the mRNA expressions of P16INK4a, HOXA9 and HOXC13 were reduced to 9.2%, 10.9% and 69.7%, respectively, as compared to control HeLa cells (P<0.01). However, hTERT and HOXB4 mRNA expressions did not change significantly (P>0.05). FACS analysis showed a decrease from 65.68 % to 50.53% in G1 population and a significant increase from 27.17% to 39.59 % in S population after transfection (P<0.01).CONCLUSION: BMI-1 over-expression in HeLa cells down-regulates mRNA expressions of P16INK4a, HOXA9 and HOXC13, decreases G1 population and increases S population. Therefore, BMI-1 may be involved in carcinogenesis and cancer development.     

Effects of shRNA targeting COL1A1 gene on proliferation and apoptosis of human breast cancer cell line MDA-MB-231

AIM: To investigate the effects of shRNA-mediated collagen type I alpha 1 (COL1A1) gene silencing on the proliferation and apoptosis of human breast cancer cell line MDA-MB-231. METHODS: The specific recombinant vector pSilencer2.1-U6-COL1A1 was transiently transfected into human breast cancer cell line MDA-MB-231 with lipofectamine. RT-PCR and Western blotting were performed to detect the expression levels of COL1A1. MTT assay was employed to evaluate the effect of COL1A1 gene silencing on the cell proliferation. Flow cytometry was used to determine the apoptosis and cell cycle of transfected cells. The morphological characteristics of apoptosis were observed by Hoechst 33258 staining. RESULTS: Compared with mock group and scrambled group, the mRNA and protein levels of COL1A1 were reduced by pshRNA-COL1A1 transfection (P<0.05). The proliferation of MDA-MB-231 cells treated in shRNA-COL1A1 was significantly inhibited in a time-dependent way. The percentages of G₀/G₁ phase cells and early apoptotic rate were significantly higher in pshRNA-COL1A1 group than those in mock and scrambled group (P<0.05). The changes of apoptotic morphology such as cell shrinkage and nuclear condensation were also observed by staining with Hoechst 33258 under fluorescence microscope. CONCLUSION: Transfection of eukaryotic expression vector pshRNA-COL1A1 effectively inhibits the proliferation, induces apoptosis and arrests MDA-MB-231 cells in G0/G1 phase.     

Regulatory effect of siRNA interference targeting BMI-1 gene on proliferation of bladder cancer EJ cells

AIM: To investigate whether siRNA-mediated BMI-1 gene silencing inhibits the proliferation of EJ cells by detecting the expression of BMI-1, p16^INK4a and p14^ARF genes at mRNA and protein levels in bladder cancer EJ cells and normal bladder transitional epithelium cells. METHODS: The protein expression and localization of BMI-1, p16^INK4a and p14^ARF in EJ cells were determined by cellular immunofluorescence. An siRNA targeting BMI-1 gene was synthesized and transfected into bladder carcinoma EJ cells by liposomes. The mRNA expression of BMI-1, p16^INK4a and p14^ARF was detected by real-time PCR and the protein levels were measured by Western blotting in bladder cancer EJ cells and normal bladder transitional epithelium cells. Cell survival was analyzed by CCK-8 assay. Cell apoptosis were examined by flow cytometry. RESULTS: The mRNA and protein expression of BMI-1 in EJ cells was higher than that in bladder transitional epithelium cells. However, the expression of p16^INK4a and p14^ARF were opposite.While BMI-1 gene in EJ cells was silenced by siRNA, the mRNA and protein expression of BMI-1 were declined whereas the expression of p16^INK4a and p14^ARF was increased. The viability of the EJ cells was decreased and the apoptotic cells were increased when BMI-1 gene was silenced. CONCLUSION: The expression of BMI-1 is inversely correlated with the expression of p16^INK4a and p14^ARF in bladder transitional cell carcinoma EJ cells. The siRNA-mediated BMI-1 gene silencing in bladder cancer EJ cells inhibits the cell growth and up-regulates the expression of p16^INK4a and p14^ARF in vitro.     

Studies on the human cytomegalovirus protein pUL23 by establishment of HELF cell line stably expressing pUL23 gene

AIM: To establish HELF cell line with stable and effective expression of human cytomegalovirus (HCMV) UL23 gene as a powerful tool to study the situation of viral proteins in host cells in vitro. METHODS: UL23 gene was amplified by PCR from HCMV and then recombinant plasmid pLEGFP-N1-FLAG-UL23 was constructed by molecular cloning techniques. Artificial retroviral viruses containing HCMV UL23 gene were obtained by transferring plasmid pLEGFP-N1-FLAG-UL23 into the AmphoPackTM-293 package cells. The HELF cells were infected by the artificial retroviral particles. The positive HELF cells which stably expressed the UL23 were obtained by G418 selection. The location of the viral proteins in the cells was observed by laser confocal microscope. RESULTS: UL23 gene was amplified by RT-PCR from the positive HELF cells, and the UL23 fusion protein was expressed correctly and confirmed by Western blotting in the positive cells, indicating that the UL23 gene was stably integrated into the chromosome of HELF cells. The viral UL23 protein expressed in HELF cells was located in the cytoplasm observed by means of laser confocal microscope. CONCLUSION: Transgene cell strain stably carrying UL23 gene is successfully constructed by retroviral gene transfer techniques. The location of HCMV pUL23 in the host cells implicates that this vial protein exerts its function in cytoplasm.