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697. Improvement of Gene Targeting at the Human rDNA Locus by Zinc Finger Nucleases
DNA VECTOROLOGY & GENE TARGETING - II ITR or the p5. Following co-transfection of HeLa cells with a chimeric RBS/trs plasmid and a Rep68 plasmid, AAVS1-specific integration of RBS/trs plasmid was detected with semi-quantitative PCR using an AAVS1-specific primer and either a transgene- or backbone-specific primer. Any RBS/trs plasmids examined were delivered into AAVS1. However, the direction of AAVS1-specific integration of RBS/trs plasmid was considerably different. Particularly the combination of the RBS and the trs sequence both from the p5 promoter sequence more tightly regulate the direction of the plasmid insertion. These findings indicate that the mechanism underlying the AAVS1-specific integration via the ITR and the p5 promoter sequence differs and provide important information for developing a gene insertion technology based on AAV integration.
clones were selected on day12. a, Clones selected after transfected with pHrn-GFP; b, clones selected after cotransfected with phrnGFP and ZFN-3; c, characterization of the selected clones by PCR. About 1.4kb band appeared in the lane corresponding to the clones underwent site-specific integration.
697. Improvement of Gene Targeting at the Human rDNA Locus by Zinc Finger Nucleases
The antitumor effect of endostatin is extensively studied and transgene has become an alternative method to deliver endostatin. To evaluate the anti-tumor efficacy of endostatin when expressed transiently and stably, a human ribosomal DNA (hrDNA) targeting vector plasmid containing recombinant human endostatin was constructed.
Youjin Hu,1 Di Xiao,1 Xionghao Liu,1 Lingqian Wu,1 Desheng Liang.1 1 State Key Laboratory of Medical Genetics, Central South University, Changsha, Hunan, China. Viral and non-viral gene delivery systems have been mainly used in gene therapy study. A Significant drawback of non-viral vector is the lack of chromosomal integration that makes it difficult to obtain sustained transgene expression, and viral random integration may have genotoxic side effects by misregulating expression of nearby genes or by disrupting cellular genes, therefore, non-viral vector has been considered as a very important alternative choice. pHrneo is a human ribosomal DNA (hrDNA) targeting vector previously constructed by our group, which can target the exogenous gene into the hrDNA locus. However, the low targeting efficiency limits its further application. Zinc finger nucleases (ZFNs) that consists of Zinc finger protein and the cleavage domain of the FokI type II restriction endonuclease can stimulate huomologous recombination significantly through creating DNA double-strand breaks (DSBs) near the target sites. In the present study, two pairs of ZFNs targeted to an endogenous site of the human rRNA gene (pHrneo target-site) were designed by modular-assemble assay. The affinity of zinc finger protein binding to the targeting site was confirmed by chromatin immunoprecipitation. The cleavage capacity at the target site was assayed by the IVTT in-vitro cleavage assay. The DSBs created by ZFNs were detected using immunofluorescence staining. The targeting efficiency of pHrneo carrying a GFP cassette was improved by ∼26 times when co-transfected with the ZFNs in the HT1080 cells.
For the first time, our findings show that the created DSBs at 1kb away from the target site can effectively stimulate the improvement of target efficiency, indicating that many more target sites can be used for improving the gene targeting by ZFNs. Figure 1. Gene targeting in HT1080 cells. After co-transfected with pHrn-GFP and ZFNs, targeted HT1080 cells were selected by adding G418, and several Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
698. Nonviral-Mediated Endostatin Delivery Results in Inhibition of Fibrosarcoma Growth
Jianxiong Peng,1,2 Lingqian Wu,1 Xionghao Liu,1 Desheng Liang.1 State Key Laboratory of Medical Genetics, Central South University, Changsha, Hunan, China; 2Faculty of Laboratory Medicine Xiangya School of Medicine, Central South University, Changsha, Hunan, China. 1
In the case of transient expression of endostatin in vivo, the plasmid mixed with liposome or amino-modified silica nanoparticles (SiNP) was injected into the implanted human fibrosarcoma (HT1080) in nude mice. In the stable expression group, the HT1080 cells with endostatin stable transgene were implanted into the mice (ex vivo). The bioactivity of the human endostatin secreted by the cells clones with stable transgene and durable expression was assayed in vitro. The expression of endostatin in vitro and in vivo was confirmed by RTPCR, western blot and immuno-histochemistry. The antiangiogenic action of endostatin was assessed by micro-vessel density stained with CD 34.
Comparing with the control, the tumor volume was reduced by 93% in stable expression group, and 56% and 42% respectively in DNASiNP and DNA-liposome group. Tumor forming rate was 35% in the S267
clones were selected on day12. a, Clones selected after transfected with pHrn-GFP; b, clones selected after cotransfected with phrnGFP and ZFN-3; c, characterization of the selected clones by PCR. About 1.4kb band appeared in the lane corresponding to the clones underwent site-specific integration.
697. Improvement of Gene Targeting at the Human rDNA Locus by Zinc Finger Nucleases
The antitumor effect of endostatin is extensively studied and transgene has become an alternative method to deliver endostatin. To evaluate the anti-tumor efficacy of endostatin when expressed transiently and stably, a human ribosomal DNA (hrDNA) targeting vector plasmid containing recombinant human endostatin was constructed.
Youjin Hu,1 Di Xiao,1 Xionghao Liu,1 Lingqian Wu,1 Desheng Liang.1 1 State Key Laboratory of Medical Genetics, Central South University, Changsha, Hunan, China. Viral and non-viral gene delivery systems have been mainly used in gene therapy study. A Significant drawback of non-viral vector is the lack of chromosomal integration that makes it difficult to obtain sustained transgene expression, and viral random integration may have genotoxic side effects by misregulating expression of nearby genes or by disrupting cellular genes, therefore, non-viral vector has been considered as a very important alternative choice. pHrneo is a human ribosomal DNA (hrDNA) targeting vector previously constructed by our group, which can target the exogenous gene into the hrDNA locus. However, the low targeting efficiency limits its further application. Zinc finger nucleases (ZFNs) that consists of Zinc finger protein and the cleavage domain of the FokI type II restriction endonuclease can stimulate huomologous recombination significantly through creating DNA double-strand breaks (DSBs) near the target sites. In the present study, two pairs of ZFNs targeted to an endogenous site of the human rRNA gene (pHrneo target-site) were designed by modular-assemble assay. The affinity of zinc finger protein binding to the targeting site was confirmed by chromatin immunoprecipitation. The cleavage capacity at the target site was assayed by the IVTT in-vitro cleavage assay. The DSBs created by ZFNs were detected using immunofluorescence staining. The targeting efficiency of pHrneo carrying a GFP cassette was improved by ∼26 times when co-transfected with the ZFNs in the HT1080 cells.
For the first time, our findings show that the created DSBs at 1kb away from the target site can effectively stimulate the improvement of target efficiency, indicating that many more target sites can be used for improving the gene targeting by ZFNs. Figure 1. Gene targeting in HT1080 cells. After co-transfected with pHrn-GFP and ZFNs, targeted HT1080 cells were selected by adding G418, and several Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
698. Nonviral-Mediated Endostatin Delivery Results in Inhibition of Fibrosarcoma Growth
Jianxiong Peng,1,2 Lingqian Wu,1 Xionghao Liu,1 Desheng Liang.1 State Key Laboratory of Medical Genetics, Central South University, Changsha, Hunan, China; 2Faculty of Laboratory Medicine Xiangya School of Medicine, Central South University, Changsha, Hunan, China. 1
In the case of transient expression of endostatin in vivo, the plasmid mixed with liposome or amino-modified silica nanoparticles (SiNP) was injected into the implanted human fibrosarcoma (HT1080) in nude mice. In the stable expression group, the HT1080 cells with endostatin stable transgene were implanted into the mice (ex vivo). The bioactivity of the human endostatin secreted by the cells clones with stable transgene and durable expression was assayed in vitro. The expression of endostatin in vitro and in vivo was confirmed by RTPCR, western blot and immuno-histochemistry. The antiangiogenic action of endostatin was assessed by micro-vessel density stained with CD 34.
Comparing with the control, the tumor volume was reduced by 93% in stable expression group, and 56% and 42% respectively in DNASiNP and DNA-liposome group. Tumor forming rate was 35% in the S267