A retroviral vector pINC-lacZ containing an E coli β-galactosidase(β-gal)gene was constructed and introduced into the MESPU-13 cells by electroporation.Four G418-resistant colonies were obtained from 1×107,elec...A retroviral vector pINC-lacZ containing an E coli β-galactosidase(β-gal)gene was constructed and introduced into the MESPU-13 cells by electroporation.Four G418-resistant colonies were obtained from 1×107,electroporated MESPU-13 cells. Histochemical staining for β-gal activity showed that lacZ gene was expressed in at least three of the four colonies.Southern analysis proved that one copy of foreign gene was integrated in the chromosome of one of the transformed lines(MC15).These results showed that the expression of lacZ gene driven by SiCMVIE promoter can be detected in the transformed MESPU-13 cells.展开更多
Objective： The aim of the study was to observe the transfection efficacy of hepatitis B virus envelope （HBVE） and evaluate its ability as a gene transfer vector for liver cancer cells. Methods： To obtain HBVE, the...Objective： The aim of the study was to observe the transfection efficacy of hepatitis B virus envelope （HBVE） and evaluate its ability as a gene transfer vector for liver cancer cells. Methods： To obtain HBVE, the supematant fluid of HepG 2.2.15 cells was mixed with a PEG8000 solution for concentration and was inactivated by β-propiolactone. The acquired HBVE was used to pack plRES2-EGFP to test its package ability. Then, we examined its quantity and quality with ELISA, PCR, SDS-PAGE and electron microscopy. The plRES2-EGFP was packed with HBVE and obtained the product HBVE-GFP. The plRES2-EGFP was packed with liposome and obtained the product liposome-GFP. HBVE-GFP and liposome-GFP were used to transfer HepG 2 cells to study the transfection efficiency. HBVE-GFP was used to transfer HepG 2, A549, HeLa and FB cells to study the targeting ability. The green fluorescent protein （GFP） expression was observed under a fluorescent microscope. The rate of GFP positive cells was determined by flow cytometry. Results： 1. The acquired HBVE could retain the surface protein HBsAg ＋ pre S1 ＋ pre S2 and had no virus DNA. It had good package ability for plRES2-EGFP. 2. Transfection efficiency： The GFP could be observed in both the liposome group and HBVE group under the fluorescent microscope. But the HBVE group had a higher fluorescent intensity than liposome group. The transfection rate of liposome group was 49.97% ＋ 2.37% while the HBVE group was 70.65% ＋ 3.15% and the fluorescent intensity of the HBVE group was 3-4 times （P = 0.000） for liposome group with the determination of flow cytometry. 3. Targeting ability： The GFP could be observed in the four groups under the fluorescent microscope. The HepG 2 group had the highest fluorescent intensity among the four groups. The transfection rate of HepG 2 group was 71.35% ＋ 0.03% which was highly expressed than other groups （P = 0.000） and the fluorescent intensity of the HepG 2 group was 2-3 times （P = 0.000） for the other 3 groups with the determination of flow cytometry. Conclusion： HBVE can be constructed successfully with the methods of PEG8000 and β-propiolactone from the supernatant fluid of HepG 22.15 cells. The HBVE can be a candidate gene transfer vector for liver cancer cells.展开更多
The cellular protein tetherin tethers the HIV-1 viral particles on the cellular membrane to inhibit the replication of HIV-1. However, the HIV-1 accessory protein Vpu counteracts the antiviral function of tetherin. In...The cellular protein tetherin tethers the HIV-1 viral particles on the cellular membrane to inhibit the replication of HIV-1. However, the HIV-1 accessory protein Vpu counteracts the antiviral function of tetherin. In this study, two retroviral vector plasmids were constructed. One inhibited the vpu gene expression; the other one over-expressed the tetherin. Both retroviral vector plasmids could be packaged in the packaging cell line PT67 to obtain the corresponding retroviruses. The retroviral vector plasmids＇ functions of tetherin over-expression or vpu-RNAi were detected at the cell level. Retroviral vector plasmids were transfected to PT67 cells at different ratios from 0T3V to 3TOV, and then mixed retroviruses were harvested. The antiviral functions of mixed retroviruses were detected in HIV-1 infected TZM-bi cells. The results showed that packaged mixed retroviruses could repress the replication of HIV-1 in TZM-bl cells.展开更多
To obtain the P8 protein of Rice gall dwarf virus (RGDV) with biological activity,its outer coat protein gene S8 was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression system.The S8...To obtain the P8 protein of Rice gall dwarf virus (RGDV) with biological activity,its outer coat protein gene S8 was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression system.The S8 gene was subcloned into the pFastBacTM1 vector,to produce the recombinant baculovirus transfer vector pFB-S8.After transformation,pFB-S8 was introduced into the competent cells (E.coli DH10Bac) containing a shuttle vector,Bacmid,generating the recombinant bacmid rbpFB-S8.After being infected by recombinant baculovirus rvpFB-S8 at different multiplicities of infection,Sf9 cells were collected at different times and analyzed by SDS-PAGE,Western blotting and immunofluorescence microscopy.The expression level of the P8 protein was highest between 48-72 h after transfection of Sf9 cells.Immunofluorescence microscopy showed that P8 protein of RGDV formed punctate structures in the cytoplasm of Sf9 cells.展开更多
Safe and efficient gene transfer systems are the basis of gene therapy applications. Non-integrating lentiviral(NIL) vectors are among the most promising candidates for gene transfer tools, because they exhibit high t...Safe and efficient gene transfer systems are the basis of gene therapy applications. Non-integrating lentiviral(NIL) vectors are among the most promising candidates for gene transfer tools, because they exhibit high transfer efficiency in both dividing and non-dividing cells and do not present a risk of insertional mutagenesis. However, non-integrating lentiviral vectors cannot introduce stable exogenous gene expression to dividing cells, thereby limiting their application. Here, we report the design of a non-integrating lentiviral vector that contains the minimal scaffold/matrix attachment region(S/MAR) sequence(SNIL), and this SNIL vector is able to retain episomal transgene expression in dividing cells. Using SNIL vectors, we detected the expression of the eGFP gene for 61 days in SNIL-transduced stable CHO cells, either with selection or not. In the NIL group without the S/MAR sequence, however, the transduced cells died under selection for the transient expression of NIL vectors. Furthermore,Southern blot assays demonstrated that the SNIL vectors were retained extrachromosomally in the CHO cells. In conclusion,the minimal S/MAR sequence retained the non-integrating lentiviral vectors in dividing cells, which indicates that SNIL vectors have the potential for use as a gene transfer tool.展开更多
Induced pluripotent stem (iPS) cell technology demonstrates that somatic cells can be reprogrammed to a pluripotent state by over-expressing four reprogramming factors.This technology has created an interest in derivi...Induced pluripotent stem (iPS) cell technology demonstrates that somatic cells can be reprogrammed to a pluripotent state by over-expressing four reprogramming factors.This technology has created an interest in deriving iPS cells from domesticated animals such as pigs,sheep and cattle.Moloney murine leukemia retrovirus vectors have been widely used to generate and study mouse iPS cells.However,this retrovirus system infects only mouse and rat cells,which limits its use in establishing iPS cells from other mammals.In our study,we demonstrate a novel retrovirus strategy to efficiently generate porcine iPS cells from embryonic fibroblasts.We transfected four human reprogramming factors (Oct4,Sox2,Klf4 and Myc) into fibroblasts in one step by using a VSV-G envelope-coated pantropic retrovirus that was easily packaged by GP2-293 cells.We established six embryonic stem (ES)-like cell lines in human ES cell medium supplemented with bFGF.Colonies showed a similar morphology to human ES cells with a high nuclei-cytoplasm ratio and phase-bright flat colonies.Porcine iPS cells could form embryoid bodies in vitro and differentiate into the three germ layers in vivo by forming teratomas in immunodeficient mice.展开更多
To study the inhibition effect of oxLDL on the uptake and clearance of intra ce llular 3H cholesterol in v SMC from the human apoA1 transgenic m ice (C57BL/6) and the changes in human apoA1 mRNA expression ...To study the inhibition effect of oxLDL on the uptake and clearance of intra ce llular 3H cholesterol in v SMC from the human apoA1 transgenic m ice (C57BL/6) and the changes in human apoA1 mRNA expression in v SMC from hum an apoA1 transgenic mice after oxLDL stimulation Methods v SMC originally isolated from human apoA1 transgenic mice connected with a rec ombined mouse metallothionein Ⅰ (MT Ⅰ) promoter was used, and the effect of oxLDL on the uptake and clearance of intracellular 3H cholesterol was s tudied in v SMC of the transgenic and control mice respectively, the study of h apoAⅠ mRNA expression from v SMC of the transgenic mice were done by RT PCR and Northern blot Results oxLDL (30?μg/ml) strongly promoted the SMC proliferation No difference was f ound in 3H cholesterol uptake between nSMC and trSMC, and the uptak e rates of both kinds of SMC rose 100% after oxLDL stimulation The efflux rate s of 3H cholesterol in trSMC were much higher than those of nSMC (40% -50%) Afte r ox LDL stimulation, the clearance rates fell by 28% and 10%, respectively, for nSMC and trSMC The result of RT PCR and Northern blot showed that h apoA1 gene e xpression was markedly increased by the stimulation of oxLDL Conclusion Expression of the h apoA1 gene in C57BL/6 mice enables them to reduce the accum ulation of cholesterol in v SMC The trSMC can alleviate the harmful effect of oxLDL due to the increase of h apoA1 expression展开更多
文摘A retroviral vector pINC-lacZ containing an E coli β-galactosidase(β-gal)gene was constructed and introduced into the MESPU-13 cells by electroporation.Four G418-resistant colonies were obtained from 1×107,electroporated MESPU-13 cells. Histochemical staining for β-gal activity showed that lacZ gene was expressed in at least three of the four colonies.Southern analysis proved that one copy of foreign gene was integrated in the chromosome of one of the transformed lines(MC15).These results showed that the expression of lacZ gene driven by SiCMVIE promoter can be detected in the transformed MESPU-13 cells.
基金Supported by a grant from the National Natural Sciences Foundation of China No 30100189
文摘Objective： The aim of the study was to observe the transfection efficacy of hepatitis B virus envelope （HBVE） and evaluate its ability as a gene transfer vector for liver cancer cells. Methods： To obtain HBVE, the supematant fluid of HepG 2.2.15 cells was mixed with a PEG8000 solution for concentration and was inactivated by β-propiolactone. The acquired HBVE was used to pack plRES2-EGFP to test its package ability. Then, we examined its quantity and quality with ELISA, PCR, SDS-PAGE and electron microscopy. The plRES2-EGFP was packed with HBVE and obtained the product HBVE-GFP. The plRES2-EGFP was packed with liposome and obtained the product liposome-GFP. HBVE-GFP and liposome-GFP were used to transfer HepG 2 cells to study the transfection efficiency. HBVE-GFP was used to transfer HepG 2, A549, HeLa and FB cells to study the targeting ability. The green fluorescent protein （GFP） expression was observed under a fluorescent microscope. The rate of GFP positive cells was determined by flow cytometry. Results： 1. The acquired HBVE could retain the surface protein HBsAg ＋ pre S1 ＋ pre S2 and had no virus DNA. It had good package ability for plRES2-EGFP. 2. Transfection efficiency： The GFP could be observed in both the liposome group and HBVE group under the fluorescent microscope. But the HBVE group had a higher fluorescent intensity than liposome group. The transfection rate of liposome group was 49.97% ＋ 2.37% while the HBVE group was 70.65% ＋ 3.15% and the fluorescent intensity of the HBVE group was 3-4 times （P = 0.000） for liposome group with the determination of flow cytometry. 3. Targeting ability： The GFP could be observed in the four groups under the fluorescent microscope. The HepG 2 group had the highest fluorescent intensity among the four groups. The transfection rate of HepG 2 group was 71.35% ＋ 0.03% which was highly expressed than other groups （P = 0.000） and the fluorescent intensity of the HepG 2 group was 2-3 times （P = 0.000） for the other 3 groups with the determination of flow cytometry. Conclusion： HBVE can be constructed successfully with the methods of PEG8000 and β-propiolactone from the supernatant fluid of HepG 22.15 cells. The HBVE can be a candidate gene transfer vector for liver cancer cells.
基金National Natural Science Foundation of China(81101245,30970162)The Fundamental Research Funds for the Central Universities(65011871)National Training Programs of Innovation for Undergraduates(111005505)
文摘The cellular protein tetherin tethers the HIV-1 viral particles on the cellular membrane to inhibit the replication of HIV-1. However, the HIV-1 accessory protein Vpu counteracts the antiviral function of tetherin. In this study, two retroviral vector plasmids were constructed. One inhibited the vpu gene expression; the other one over-expressed the tetherin. Both retroviral vector plasmids could be packaged in the packaging cell line PT67 to obtain the corresponding retroviruses. The retroviral vector plasmids＇ functions of tetherin over-expression or vpu-RNAi were detected at the cell level. Retroviral vector plasmids were transfected to PT67 cells at different ratios from 0T3V to 3TOV, and then mixed retroviruses were harvested. The antiviral functions of mixed retroviruses were detected in HIV-1 infected TZM-bi cells. The results showed that packaged mixed retroviruses could repress the replication of HIV-1 in TZM-bl cells.
基金supported by the National Science Foundation of China (30970135)The Key Project of Genetically Modified Organisms Breeding(2009ZX08009-044B)+1 种基金the Natural Science Foundation of Fujian Province of China (No.2006J0065)the Public-interest Scientific Institution Basal Research Fund of Fujian Province (2009R10029-3)
文摘To obtain the P8 protein of Rice gall dwarf virus (RGDV) with biological activity,its outer coat protein gene S8 was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression system.The S8 gene was subcloned into the pFastBacTM1 vector,to produce the recombinant baculovirus transfer vector pFB-S8.After transformation,pFB-S8 was introduced into the competent cells (E.coli DH10Bac) containing a shuttle vector,Bacmid,generating the recombinant bacmid rbpFB-S8.After being infected by recombinant baculovirus rvpFB-S8 at different multiplicities of infection,Sf9 cells were collected at different times and analyzed by SDS-PAGE,Western blotting and immunofluorescence microscopy.The expression level of the P8 protein was highest between 48-72 h after transfection of Sf9 cells.Immunofluorescence microscopy showed that P8 protein of RGDV formed punctate structures in the cytoplasm of Sf9 cells.
基金supported by the Major State Basic Research Development Program of China(2011CB965203)
文摘Safe and efficient gene transfer systems are the basis of gene therapy applications. Non-integrating lentiviral(NIL) vectors are among the most promising candidates for gene transfer tools, because they exhibit high transfer efficiency in both dividing and non-dividing cells and do not present a risk of insertional mutagenesis. However, non-integrating lentiviral vectors cannot introduce stable exogenous gene expression to dividing cells, thereby limiting their application. Here, we report the design of a non-integrating lentiviral vector that contains the minimal scaffold/matrix attachment region(S/MAR) sequence(SNIL), and this SNIL vector is able to retain episomal transgene expression in dividing cells. Using SNIL vectors, we detected the expression of the eGFP gene for 61 days in SNIL-transduced stable CHO cells, either with selection or not. In the NIL group without the S/MAR sequence, however, the transduced cells died under selection for the transient expression of NIL vectors. Furthermore,Southern blot assays demonstrated that the SNIL vectors were retained extrachromosomally in the CHO cells. In conclusion,the minimal S/MAR sequence retained the non-integrating lentiviral vectors in dividing cells, which indicates that SNIL vectors have the potential for use as a gene transfer tool.
基金supported by the National Basic Research Program of China (Grant Nos. 2009CB941003, 2011CBA0110 and 2011CBA01000)
文摘Induced pluripotent stem (iPS) cell technology demonstrates that somatic cells can be reprogrammed to a pluripotent state by over-expressing four reprogramming factors.This technology has created an interest in deriving iPS cells from domesticated animals such as pigs,sheep and cattle.Moloney murine leukemia retrovirus vectors have been widely used to generate and study mouse iPS cells.However,this retrovirus system infects only mouse and rat cells,which limits its use in establishing iPS cells from other mammals.In our study,we demonstrate a novel retrovirus strategy to efficiently generate porcine iPS cells from embryonic fibroblasts.We transfected four human reprogramming factors (Oct4,Sox2,Klf4 and Myc) into fibroblasts in one step by using a VSV-G envelope-coated pantropic retrovirus that was easily packaged by GP2-293 cells.We established six embryonic stem (ES)-like cell lines in human ES cell medium supplemented with bFGF.Colonies showed a similar morphology to human ES cells with a high nuclei-cytoplasm ratio and phase-bright flat colonies.Porcine iPS cells could form embryoid bodies in vitro and differentiate into the three germ layers in vivo by forming teratomas in immunodeficient mice.
文摘To study the inhibition effect of oxLDL on the uptake and clearance of intra ce llular 3H cholesterol in v SMC from the human apoA1 transgenic m ice (C57BL/6) and the changes in human apoA1 mRNA expression in v SMC from hum an apoA1 transgenic mice after oxLDL stimulation Methods v SMC originally isolated from human apoA1 transgenic mice connected with a rec ombined mouse metallothionein Ⅰ (MT Ⅰ) promoter was used, and the effect of oxLDL on the uptake and clearance of intracellular 3H cholesterol was s tudied in v SMC of the transgenic and control mice respectively, the study of h apoAⅠ mRNA expression from v SMC of the transgenic mice were done by RT PCR and Northern blot Results oxLDL (30?μg/ml) strongly promoted the SMC proliferation No difference was f ound in 3H cholesterol uptake between nSMC and trSMC, and the uptak e rates of both kinds of SMC rose 100% after oxLDL stimulation The efflux rate s of 3H cholesterol in trSMC were much higher than those of nSMC (40% -50%) Afte r ox LDL stimulation, the clearance rates fell by 28% and 10%, respectively, for nSMC and trSMC The result of RT PCR and Northern blot showed that h apoA1 gene e xpression was markedly increased by the stimulation of oxLDL Conclusion Expression of the h apoA1 gene in C57BL/6 mice enables them to reduce the accum ulation of cholesterol in v SMC The trSMC can alleviate the harmful effect of oxLDL due to the increase of h apoA1 expression
