Establishment of leukemia cell model with inducible AML1-ETO expression and its effect on fatty acid metabolism in leukemia cells / 中华血液学杂志

Objective: To investigate the effect of the AML1-ETO (AE) fusion gene on the biological function of U937 leukemia cells by establishing a leukemia cell model that induces AE fusion gene expression. Methods: The doxycycline (Dox) -dependent expression of the AE fusion gene in the U937 cell line (U937-AE) were established using a lentivirus vector system. The Cell Counting Kit 8 methods, including the PI and sidanilide induction, were used to detect cell proliferation, cell cycle-induced differentiation assays, respectively. The effect of the AE fusion gene on the biological function of U937-AE cells was preliminarily explored using transcriptome sequencing and metabonomic sequencing. Results: ①The Dox-dependent Tet-on regulatory system was successfully constructed to regulate the stable AE fusion gene expression in U937-AE cells. ②Cell proliferation slowed down and the cell proliferation rate with AE expression (3.47±0.07) was lower than AE non-expression (3.86 ± 0.05) after inducing the AE fusion gene expression for 24 h (P<0.05). The proportion of cells in the G(0)/G(1) phase in the cell cycle increased, with AE expression [ (63.45±3.10) %) ] was higher than AE non-expression [ (41.36± 9.56) %] (P<0.05). The proportion of cells expressing CD13 and CD14 decreased with the expression of AE. The AE negative group is significantly higher than the AE positive group (P<0.05). ③The enrichment analysis of the transcriptome sequencing gene set revealed significantly enriched quiescence, nuclear factor kappa-light-chain-enhancer of activated B cells, interferon-α/γ, and other inflammatory response and immune regulation signals after AE expression. ④Disorder of fatty acid metabolism of U937-AE cells occurred under the influence of AE. The concentration of the medium and short-chain fatty acid acylcarnitine metabolites decreased in cells with AE expressing, propionyl L-carnitine, wherein those with AE expression (0.46±0.13) were lower than those with AE non-expression (1.00±0.27) (P<0.05). The metabolite concentration of some long-chain fatty acid acylcarnitine increased in cells with AE expressing tetradecanoyl carnitine, wherein those with AE expression (1.26±0.01) were higher than those with AE non-expression (1.00±0.05) (P<0.05) . Conclusion: This study successfully established a leukemia cell model that can induce AE expression. The AE expression blocked the cell cycle and inhibited cell differentiation. The gene sets related to the inflammatory reactions was significantly enriched in U937-AE cells that express AE, and fatty acid metabolism was disordered.

The Cre-loxP system and its derivative systems: methodology research and applications in neuroscience / 药学学报

In scientific research, it is often needed to knock in, knock out, knock down, or overexpress a specific gene in model organisms or specific types of cells to achieve precise regulation of experimental independent variables. In this case, various transgenic mice are required. The cyclization recombinase (Cre) can directly interact with different loxP (locus X over P1) DNA sequences without any cofactors to perform specific gene knock-in or knock-out at specific targets. Because of its advantages of simple action principles, high spatial specificity, and high reorganization efficiency, the Cre-loxP system is widely used in scientific research. Furthermore, the CreERT2 system (mutant of the fusion protein of Cre and estrogen receptor ligand binding domain) and the tetracycline (Tet)-on/off system, derived from the Cre-loxP system, have made the recombination of the target gene occur in temporal-specificity on the basis of spatial-specificity. This dual specificity of time and space is indispensable for research in specific directions such as fear memory and engram cells on the basis of reducing the impacts on experimental animals. Therefore, these derived systems have broad application prospects.

Suppressive effect of Tet-on mediated livin RNA interference on growth of lung carcinoma xenegraft in a rat model / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To investigate he effect of tetracycline- (Tet-on) mediated livin RNA interference on growth of lung carcinoma xenegrafts, and find a better regulatory way to interfere the development on lung cancer. Methods: livin shRNA lentiviral vectors were constructed; and the lung cancerA549 cells were subcutaneously injected into right upper back of nude mice to establish xenegraft model. The livin shRNAlentiviral vectors were injected into xenografts to interfere the expression of livin, then tetracycline was injected intraperitoneally for the induction. The suppressive effect of Tet-on mediated livin RNA interference efficiency was investigated and lung cancer xenograft development was observed. Results: After the induction with Tet-on, livin gene expression was significantly inhibited by livin shRNAcompared with the control group and Tet-on-NC group; the xenograft volume in Tet-on- livin shRNAgroup was significantly smaller than that in control group and Tet-on-NC group ([5.31±0.86]g vs [8.22±0.63]g and [7.17±0.54] g, P<0.05). Moreover, little body toxicity was observed and no nude mice died in this study. Conclusion: The Tet-on mediated livin shRNA could suppress the growth of lung cancer development with good targeting and controllable characteristics, which might provide a potent tool for treating lung cancer with livin protein as target.

Pin1 expression in the skin and establishment of an inducible transgenic mouse model / 中国实验动物学报

Objective To observe the Pin1 expression pattern in skin and to establish an inducible skin specific Pin1 overexpression mouse model. Methods The mouse Pin1 gene was cloned into modified vector pTRE2 with C?terminal Myc tag. The linearized pTRE2?Pin1 DNA was micro?injected into one?cell embryos followed by implantation into foster mice to produce TRE?Pin1 transgenic mice. Results TRE?Pin1 transgenic founder mice were successfully created. These mice were crossed with transgenic tool mice K14?rtTA to create epithelial specific double transgenic progenies. Pin1 gene was induced by incorporating doxycycline into drinking water of the mice. Pin1 protein overexpression in the skin was con?firmed by Western blot and immunohistochemistry. The endogenous Pin1 protein was predominantly expressed in epidermal cells in the skin. Conclusions The inducible skin specific Pin1 overexpression mouse model is successfully established which may serve as a useful model for further study of Pin1 functions in the skin.

Construction of HaCaT cell lines stably expressing the human GJB6 gene by using a Tet-On lentiviral vector and their identification / 中华皮肤科杂志

Objective To construct HaCaT cell lines stably expressing the wild type human GJB6 gene or its mutant by using a Tet-On lentiviral vector, and to lay an experimental foundation for studies on pathogenesis of hidrotic ectodermal dysplasia. Methods The wild-type human GJB6 gene and its mutant (A88V)were amplified by PCR, and then inserted into the Tet-on lentivirus plasmid to construct recombinant lentivirus vectors. The recombinants were identified by gene sequencing and enzymatic digestion. Cultured HaCaT cells were classified into three groups to be transfected with a negative control lentiviral vector (NC group), the lentivirus vector expressing the wild-type human GJB6 gene (WT group), or the lentivirus vector expressing the mutant human GJB6 gene (MU group). Puromycin was used to select HaCaT cell clones stably expressing the GJB6 gene which encodes the connexin 30 (Cx30)protein. The selected HaCaT cell clones were cultured with or without tetracycline for 48 hours, thereafter, real-time PCR(RT-PCR) was performed to detect GJB6 gene mRNA expression, Western-blot analysis to measure expressions of Cx30 and FLAG-tag proteins, and cell counting kit 8 (CCK8)assay to evaluate cellular proliferative activity. Results Enzymatic digestion and gene sequencing showed that recombinant lentivirus plasmids were successfully constructed. RT-PCR showed evidently increased mRNA expression of the GJB6 gene in stably transfected HaCaT cells. Moreover, the expression abundance of the GJB6 gene was 112.369 times higher in the WT group induced by tetracycline than in that without tetracycline treatment (P 0.05). Conclusion HaCaT cell lines which stably express the wild-type GJB6 gene or its mutant(A88V)are successfully constructed.

Experimental study on expression of TH and GDNF bi-gene of lentivirus by improved Tet-On system for a rat model of Parkinson’ s disease / 中国药理学通报

Aim To investigate TH and GDNF genes expression and regulation of lentivirus ( Lv-TH-GDNF ) based on improved Tet-On system and the effect of the Lv-TH-GDNF intrastriatal transfer on a rat Parkinson’ s disease( PD) model. Methods 1. HeLa cells were infected by obtained Lv-TH-GDNF and rtTA2 s-M2 vi-rus. The expression of tyrosine hydroxylase ( TH ) and glial cell line-derived neurotrophic factor( GDNF) genes was induced by doxycycline( Dox) which was examined by Western blot. 2. The Lv-TH-GDNF together with rtTA2 s-M2 viruses were injected into lesion-side stria-tum of a rat PD model, and the expression of GDNF and TH genes was induced by Dox. Then, the effects of Lv-TH-GDNF were evaluated by the apomorphine-induced rotational behavior, the number of dopaminer-gic neurons in substantia nigra,DA and DOPAC levels in the lesion-side striatum. In addition, Western blot was performed to check the expression of TH and GD-NF genes in the transplanted striatum. Results 1. In vitro studies on HeLa cells, Western blot showed clear protein bands of TH and GDNF in the Dox-positive group, but not in the Dox-negative group. 2. In vivo experiments in animals, the results showed that, 4 weeks after transplantation, the apomorphine-induced turning effect was significantly improved ( P<0 . 01 ) , the number of TH-positive cells in the lesion-side sub-stantia nigra pars compacta as well as the content of DA and DOPAC, the protein level of GDNF and TH genes in the lesion-side striatum was significantly in-creased ( P<0 . 01 ) , each of which was only in Lv-TH-GDNF+rtTA2 s-M2+Dox-treated rats as compared with PBS-treated rats. Conclusion The expression of TH and GDNF genes in Lv-TH-GDNF based on im-proved Tet-On system is effectively regulated by tetra-cycline antibiotics without basal activity in vitro, and the intrastriatal transfer of which has certain therapeutic effect on PD rats.

STGC3 inhibits xenograft tumor growth of nasopharyngeal carcinoma cells by altering the expression of proteins associated with apoptosis

STGC3 is a potential tumor suppressor that inhibits the growth of the nasopharyngeal carcinoma cell line CNE2; the expression of this protein is reduced in nasopharyngeal carcinoma compared with normal nasopharyngeal tissue. In this study, we investigated the tumor-suppressing activity of STGC3 in nude mice injected subcutaneously with Tet/pTRE-STGC3/CNE2 cells. STGC3 expression was induced by the intraperitoneal injection of doxycycline (Dox). The volume mean of Tet/pTRE-STGC3/CNE2+Dox xenografts was smaller than that of Tet/pTRE/CNE2+Dox xenografts. In addition, Tet/pTRE-STGC3/CNE2+Dox xenografts showed an increase in the percentage of apoptotic cells, a decrease in Bcl-2 protein expression and an increase in Bax protein expression. A proteomic approach was used to assess the protein expression profile associated with STGC3-mediated apoptosis. Western blotting confirmed the differential up-regulation of prohibitin seen in proteomic analysis. These results indicate that overexpression of STGC3 inhibits xenograft growth in nude mice by enhancing apoptotic cell death through altered expression of apoptosis-related proteins such as Bcl-2, Bax and prohibitin. These data contribute to our understanding of the function of STGC3 in human nasopharyngeal carcinoma and provide new clues for investigating other STGC3-associated tumors.

Effects of HIF-1α expression regulated by Tet-on gone expression system on hepatoma cell proliferation and cell cycle in vitro / 中华普通外科杂志

Objective To investigate the effects of HIF-1α expression regulated by Tet-on gene expression system on cell proliferation and cell cycle of hepatoma cells in vitro. Methods The change of human hepatocellular carcinoma cell lines HepG2 cell cycle and cell proliferation was measured after HIF-1 α expression of HepG2 in vitro was regulated by Tet-on expression system. Results Amplified products were confirmed as the cDNA of HIF-1α by DNA sequencing, and pTRE-HIF-1α obtained by edonuclease digestion,capable of expression in HepG2 Tet-on cells. After being incubated under different concentrations of doxycycline for 48 h, MTT assays showed that up-regulation of HIF-1α expression increased HepG2 cell proliferation activities. The cell index of S and G2/M phase was significantly higher and that of G0/G1 phase reduced with the increasing concentrations of doxycycline. The mRNA expression of Cyclin A increased with the increasing concentrations of doxycycline ( P ＜ 0. 001 ), CyclinD1 and CyclinE did not change ( P ＞0. 05). Conclusion HIF-1 α gene promotes cell proliferation and cell cycle of hcpatoma cells in vitro, and this effects increased with the increasing of HIF-1α expression possibly through influencing the expression of CyclinA.

Tumorigenicity Investigation of CNE2 Cell Line STGC3 Expression Induced by Tet-on System in Nude Mice / 生物化学与生物物理进展

STGC3, a novel tumor related gene, was cloned recently. The previous studies indicated that STGC3 can inhibit the proliferation of CNE2 cell line in vitro. To examine the effect of STGC3 on the tumorigenicity of CNE2 cell line and explore its mechanism in nude mice. The Tet/pTRE/CNE2-STGC3 cell line was planted under the front leg skin of nude mice and induced by doxycycline (Dox). The mRNA and protein level of STGC3 in transplanted tumor tissues were detected with RT-PCR and Western Blotting. The apoptosis ratio of the tumor cell was analyzed with flow cytometry. STGC3, Bcl-2 and Bax proteins were examined by immunohistochemistry method. The results indicated that high level of STGC3 expression can inhibit tumorigenicity of CNE2 cell line in nude mice. Tumor grew slowly, later and smaller. Cell apoptotic percentage increased. Bcl-2 protein expression was down-regulated and Bax protein expression was up-regulated in Tet/pTRE/CNE2-STGC3 cell line (P

Tet Regulating Expression System Establishment and Functional Analysis ofNovel Gene STGC3 in Nasopharyngeal Carcionma Cell Line CNE2 / 生物化学与生物物理进展

In an attampt to establish the functional expression of STGC3 with doxycycline (Dox) induced Tet-onregulating system in human nasopharyngeal carcinoma cell line CNE2, an ideal experimental platform wasprovided for further studies of STGC3. pTet-on regulating plasmid was transfected into CNE2, and stableexpression of Tet-on was established in CNE2 through G418 select. Then the response plasmid of recombinantpTRE-STGC3 was steadily transfected into positive CNE2/Tet-on cells with hygromycin screen. Dox was used toinduce the expression of STGC3 and a cell clone sensitive to Dox was selected. The best-induced concentrationwas determined with different concentration of Dox induction. Growth curves, clone formation rate and cell cycledistribution were detected after STGC3 gene up-regulated expression with Dox induction. The growth capacity andclone formation potential of CNE2/Tet /pTRE-STGC3 was significantly suppressed, compared with the controls(P

Advances of Lentiviral Vectors Design and Application / 中国生物工程杂志

As an efficient gene transfer vehicle lentiviral vector has been widely used in therapy research. Comparing with other retrovirus vectors, lentiviral vectors have the unique ablility of transfecting nondividing cells and terminal differentiated cells. In addition, lentiviral vectors can accommodate two or more promoters and can carry larger foreign gene insertions. Now the new generation of lentiviral vectors encoding transcriptional control sequence provides effective means for the regulation of foreign gene expression. The development of lentiviral vectors and its application in the gene therapy field were summarized.

The effect of Tet-on gene regulated HIF-1?expression on hepatoma cell apoptosis in vitro / 中国普通外科杂志

Objective To investigate the effects of Tet-on gene regulated HIF-1?expression on cell apoptosis of hepatoma cells in vitro. Methods The apoptosis of hepatocellular carcinoma cell lines HePG2 was measured after HIF-1?expression was activated in HePG2 in vitro by Tet-on expression system. Results The amplified products were confirmed as the cDNA of HIF-1?by DNA sequencing, and obtained pTHE-HIF-1?was verified by endonuclease digestion. The HePG2Tet-on cells express HIF-1?constantly. After being incubated under different concentrations of doxycycline for 48 h, HIF-1?gene could inhibit HePG2 cell apoptosis. The apoptosis rates were 59. 6%、50. 9%、38. 1%、30. 5%、23. 9%、18. 3% respectively when the concentrations of doxycycline were 0?g/ml,0. 02?g/ml,0. 2?g/ml、1?g/ml、2?g/ml,5?g/ml (P

Establishment of HepG2~(Tet-on) cell line controlled by the Tet-on regulatory system / 中国普通外科杂志

Objective To establish tetracycline-controlled inducible system(Tet-on) in HepG2 cell for further research of the function of related gens.Methods The HepG2 cells were transfected with pWHE146 vector by using liposome transfection reagent.The transfected cells were selected in medium containing G418,and G418-resistant clones were isolated.All individual G418-resistant clones were screened by transient transfection with plasmid pTRE-hyg-luc for clones with low background and high induction of luciferase in response to Dox.Results One HepG2 cell line,which exhibited high levels of induction(154,106 times)and high gene expression levels,was obtained.Conclusions The HepG2 cell line can be used to highly express eukaryotic gene and this Tet-on system is available for use in eukaryotic gene function studies.