The production of cells capable of expressing gene(s) of interest is important for a variety of applications in biomedicine and biotechnology, including gene therapy and a novel method of stem cell therapy in the vari...The production of cells capable of expressing gene(s) of interest is important for a variety of applications in biomedicine and biotechnology, including gene therapy and a novel method of stem cell therapy in the various diseases. Achieving high levels of transgene expression for the longer period of time, without adversely affecting cell viability and differentiation capacity of the cells, is crucial. In the present study, we investigated the efficiency of plasmid vector for the production of transgenic cMSCs and examined any functional change of cells after transfection. To do so first we have collected bone marrows from the adult goats and cultured them for isolation of mesenchymal stem cells (cBM-MSCs). These cells were characterized using MSC specific markers including differentiation into osteocytes and adipocytes. Transfection with plasmid vector did not adversely affect cBM-MSCs morphology, viability or differentiation potential, and transgene expression levels were unaffected beyond passage 12th. The results indicated that we have been able to generate transgenic caprine MSC (tcBM-MSC) and transfection of cBM-MSCs using plasmid vector resulted in very high and stable transfection efficiency. This finding may have considerable significance in improving the efficacy of MSC-based therapies and their tracking in animal model.展开更多
This review chronicles the development of the plant binary vectors of Ti plasmid in Agrobacterium tumefaciens during the last 30 years. A binary vector strategy was designed in 1983 to separate the T-DNA region in a s...This review chronicles the development of the plant binary vectors of Ti plasmid in Agrobacterium tumefaciens during the last 30 years. A binary vector strategy was designed in 1983 to separate the T-DNA region in a small plasmid from the virulence genes in avirulent T-DNA-less Ti plasmid. The small plant vectors with the T-DNA region have been simply now called binary Ti vectors. A binary Ti vector consist of a broad host-range replicon for propagation in A. tumeraciens, an antibiotic resistance gene for bacterial selection and the T-DNA region that would be transferred to the plant genome via the bacterial virulence machinery. The T-DNA region delimited by the right and left border sequences contains an antibiotic resistance gene for plant selection, reporter gene, and/or any genes of interest. The ColEI replicon was also added to the plasmid backbone to enhance the propagation in Escherichia coli. A general trend in the binary vector development has been to increase the plasmid stability during a long co-cultivation period of A. tumefaciens with the target host plant tissues. A second trend is to understand the molecular mechanism of broad host-range replication, and to use it to reduce the size of plasmid for ease in cloning and for higher plasmid yield in E. coli. The broad host-range replicon of VS1 was shown to be a choice of replicon over those of pRK2, pRi and pSA because of the superior stability and of small well-defined replicon. Newly developed plant binary vectors pLSU has the small size of plasmid backbone (4566 bp) consisting of VS1 replicon (2654 bp), ColE1 replicon (715 bp), a bacterial kanamycin (999 bp) or tetracycline resistance gene, and the T-DNA region (152 bp).展开更多
Saccharolobus islandicus REY15A represents one of the very few archaeal models with versatile genetic tools,which include efficient genome editing,gene silencing,and robust protein expression systems.However,plasmid v...Saccharolobus islandicus REY15A represents one of the very few archaeal models with versatile genetic tools,which include efficient genome editing,gene silencing,and robust protein expression systems.However,plasmid vectors constructed for this crenarchaeon thus far are based solely on the pRN2 cryptic plasmid.Although this plasmid coexists with pRN1 in its original host,early attempts to test pRN1-based vectors consistently failed to yield any stable host-vector system for Sa.islandicus.We hypothesized that this failure could be due to the occurrence of CRISPR immunity against pRN1 in this archaeon.We identified a putative target sequence in orf904 encoding a putative replicase on pRN1(target N1).Mutated targets(N1a,N1b,and N1c)were then designed and tested for their capability to escape the host CRISPR immunity by using a plasmid inter-ference assay.The results revealed that the original target triggered CRISPR immunity in this archaeon,whereas all three mutated targets did not,indicating that all the designed target mutations evaded host immunity.These mutated targets were then incorporated into orf904 individually,yielding corresponding mutated pRN1 backbones with which shuttle plasmids were constructed(pN1aSD,pN1bSD,and pN1cSD).Sa.islandicus transformation revealed that pN1aSD and pN1bSD were functional shuttle vectors,but pN1cSD lost the capability for replication.These results indicate that the missense mutations in the conserved helicase domain in pN1c inactivated the replicase.We further showed that pRN1-based and pRN2-based vectors were stably maintained in the archaeal cells either alone or in combination,and this yielded a dual plasmid system for genetic study with this important archaeal model.展开更多
AIM: To construct a recombinant murine CD40 ligand (mCD40L) eukaryotic expression vector for gene therapy and target therapy of hepatocellular carcinoma (HCC).METHODS: mCD40L cDNA was synthesized by RT-PCR with the sp...AIM: To construct a recombinant murine CD40 ligand (mCD40L) eukaryotic expression vector for gene therapy and target therapy of hepatocellular carcinoma (HCC).METHODS: mCD40L cDNA was synthesized by RT-PCR with the specific primers and directly cloned into T vector to generate middle recombinant. After digestion with restriction endonuclease, the target fragment was subcloned into the multi-clone sites of the eukaryotic vector. The constructed vector was verified by enzyme digestion and sequencing,and the product expressed was detected by RT-PCR and immunofluorescence methods.RESULTS: The full-length mCD40L-cDNA was successfully cloned into the eukaryotic vector through electrophoresis,and mCD40L gene was integrated into the genome of infected H22 cells by RT-PCR. Murine CD40L antigen molecule was observed in the plasma of mCD40L-H22 by indirect immuno-fluorescence staining.CONCLUSION: The recombined mCD40L eukaryotic expression vector can be expressed in H22 cell line. It providesexperimental data for gene therapy and target therapy ofhepatocellular carcinoma.展开更多
Objective: To study the therapeutic potential of Fas inhibition in different diseases, a Fas-targeting siRNA (small interfering)-expressing plasmid was constructed. Methods: The U6 promoter cassette and siFas (small i...Objective: To study the therapeutic potential of Fas inhibition in different diseases, a Fas-targeting siRNA (small interfering)-expressing plasmid was constructed. Methods: The U6 promoter cassette and siFas (small interfering RNA that inhibit Fas expression) template sequence were obtained by PCR method. They were cloned into modified pcDNA3.1. The resultant plasmid pU6-siFas was transfected into P815 cells with lipofectin2000 and selected under G-418-containing culture medium. Fas inhibition in stably transfected cells was detected by immunocytochemistry. Results: The plasmid pU6-siFas efficiently reduced the expression of Fas and conferred G-418 resistance in P815 cells. Conclusion: The successful construction of the siRNA expressing plasmid will facilitate the application of RNA interference technique and lay the foundation for further study of Fas inhibition in the treatment of different diseases such as aplastic anemia and acute liver failure.展开更多
By employing the pUC19 as a backbone,the regulatory and signal sequences which encode kanamycin resistance, and mycobacterial plasmid origin of replication (oriM) were cloned into the pUC19. The recombinant E. Coli-my...By employing the pUC19 as a backbone,the regulatory and signal sequences which encode kanamycin resistance, and mycobacterial plasmid origin of replication (oriM) were cloned into the pUC19. The recombinant E. Coli-mycobacteria shuttle expression plasmid PBCG-8000 was constructed. The PBCG-8000 was able to replicate in both E. Coli and mycobacteria (including BCG) systems, and to confer stable kanamycin resistance upon transformants. The study should facilitate the development of BCG and other mycobacteria into multivalent vaccine vectors.展开更多
文摘The production of cells capable of expressing gene(s) of interest is important for a variety of applications in biomedicine and biotechnology, including gene therapy and a novel method of stem cell therapy in the various diseases. Achieving high levels of transgene expression for the longer period of time, without adversely affecting cell viability and differentiation capacity of the cells, is crucial. In the present study, we investigated the efficiency of plasmid vector for the production of transgenic cMSCs and examined any functional change of cells after transfection. To do so first we have collected bone marrows from the adult goats and cultured them for isolation of mesenchymal stem cells (cBM-MSCs). These cells were characterized using MSC specific markers including differentiation into osteocytes and adipocytes. Transfection with plasmid vector did not adversely affect cBM-MSCs morphology, viability or differentiation potential, and transgene expression levels were unaffected beyond passage 12th. The results indicated that we have been able to generate transgenic caprine MSC (tcBM-MSC) and transfection of cBM-MSCs using plasmid vector resulted in very high and stable transfection efficiency. This finding may have considerable significance in improving the efficacy of MSC-based therapies and their tracking in animal model.
文摘This review chronicles the development of the plant binary vectors of Ti plasmid in Agrobacterium tumefaciens during the last 30 years. A binary vector strategy was designed in 1983 to separate the T-DNA region in a small plasmid from the virulence genes in avirulent T-DNA-less Ti plasmid. The small plant vectors with the T-DNA region have been simply now called binary Ti vectors. A binary Ti vector consist of a broad host-range replicon for propagation in A. tumeraciens, an antibiotic resistance gene for bacterial selection and the T-DNA region that would be transferred to the plant genome via the bacterial virulence machinery. The T-DNA region delimited by the right and left border sequences contains an antibiotic resistance gene for plant selection, reporter gene, and/or any genes of interest. The ColEI replicon was also added to the plasmid backbone to enhance the propagation in Escherichia coli. A general trend in the binary vector development has been to increase the plasmid stability during a long co-cultivation period of A. tumefaciens with the target host plant tissues. A second trend is to understand the molecular mechanism of broad host-range replication, and to use it to reduce the size of plasmid for ease in cloning and for higher plasmid yield in E. coli. The broad host-range replicon of VS1 was shown to be a choice of replicon over those of pRK2, pRi and pSA because of the superior stability and of small well-defined replicon. Newly developed plant binary vectors pLSU has the small size of plasmid backbone (4566 bp) consisting of VS1 replicon (2654 bp), ColE1 replicon (715 bp), a bacterial kanamycin (999 bp) or tetracycline resistance gene, and the T-DNA region (152 bp).
基金funded by the National Key R&D Program of China(Grant No.2020YFA0906800 to Q.S.)the National Natural Science Foundation of China(Nos.32270040 to Q.S.,32001022 to X.F.,and 32370033 to Y.S.).
文摘Saccharolobus islandicus REY15A represents one of the very few archaeal models with versatile genetic tools,which include efficient genome editing,gene silencing,and robust protein expression systems.However,plasmid vectors constructed for this crenarchaeon thus far are based solely on the pRN2 cryptic plasmid.Although this plasmid coexists with pRN1 in its original host,early attempts to test pRN1-based vectors consistently failed to yield any stable host-vector system for Sa.islandicus.We hypothesized that this failure could be due to the occurrence of CRISPR immunity against pRN1 in this archaeon.We identified a putative target sequence in orf904 encoding a putative replicase on pRN1(target N1).Mutated targets(N1a,N1b,and N1c)were then designed and tested for their capability to escape the host CRISPR immunity by using a plasmid inter-ference assay.The results revealed that the original target triggered CRISPR immunity in this archaeon,whereas all three mutated targets did not,indicating that all the designed target mutations evaded host immunity.These mutated targets were then incorporated into orf904 individually,yielding corresponding mutated pRN1 backbones with which shuttle plasmids were constructed(pN1aSD,pN1bSD,and pN1cSD).Sa.islandicus transformation revealed that pN1aSD and pN1bSD were functional shuttle vectors,but pN1cSD lost the capability for replication.These results indicate that the missense mutations in the conserved helicase domain in pN1c inactivated the replicase.We further showed that pRN1-based and pRN2-based vectors were stably maintained in the archaeal cells either alone or in combination,and this yielded a dual plasmid system for genetic study with this important archaeal model.
基金Supported by the Public Health Department Foundation of Hunan province, China, No. Y02-42
文摘AIM: To construct a recombinant murine CD40 ligand (mCD40L) eukaryotic expression vector for gene therapy and target therapy of hepatocellular carcinoma (HCC).METHODS: mCD40L cDNA was synthesized by RT-PCR with the specific primers and directly cloned into T vector to generate middle recombinant. After digestion with restriction endonuclease, the target fragment was subcloned into the multi-clone sites of the eukaryotic vector. The constructed vector was verified by enzyme digestion and sequencing,and the product expressed was detected by RT-PCR and immunofluorescence methods.RESULTS: The full-length mCD40L-cDNA was successfully cloned into the eukaryotic vector through electrophoresis,and mCD40L gene was integrated into the genome of infected H22 cells by RT-PCR. Murine CD40L antigen molecule was observed in the plasma of mCD40L-H22 by indirect immuno-fluorescence staining.CONCLUSION: The recombined mCD40L eukaryotic expression vector can be expressed in H22 cell line. It providesexperimental data for gene therapy and target therapy ofhepatocellular carcinoma.
基金Project (No. 20012131) supported by the National Administer of Health-supported Clinical Department Developing Funding, China
文摘Objective: To study the therapeutic potential of Fas inhibition in different diseases, a Fas-targeting siRNA (small interfering)-expressing plasmid was constructed. Methods: The U6 promoter cassette and siFas (small interfering RNA that inhibit Fas expression) template sequence were obtained by PCR method. They were cloned into modified pcDNA3.1. The resultant plasmid pU6-siFas was transfected into P815 cells with lipofectin2000 and selected under G-418-containing culture medium. Fas inhibition in stably transfected cells was detected by immunocytochemistry. Results: The plasmid pU6-siFas efficiently reduced the expression of Fas and conferred G-418 resistance in P815 cells. Conclusion: The successful construction of the siRNA expressing plasmid will facilitate the application of RNA interference technique and lay the foundation for further study of Fas inhibition in the treatment of different diseases such as aplastic anemia and acute liver failure.
文摘By employing the pUC19 as a backbone,the regulatory and signal sequences which encode kanamycin resistance, and mycobacterial plasmid origin of replication (oriM) were cloned into the pUC19. The recombinant E. Coli-mycobacteria shuttle expression plasmid PBCG-8000 was constructed. The PBCG-8000 was able to replicate in both E. Coli and mycobacteria (including BCG) systems, and to confer stable kanamycin resistance upon transformants. The study should facilitate the development of BCG and other mycobacteria into multivalent vaccine vectors.
