The current method for combining transgenes into a genome is through the assortment of independent loci, a classical operating system compatible with transgenic traits created by different developers, at different tim...The current method for combining transgenes into a genome is through the assortment of independent loci, a classical operating system compatible with transgenic traits created by different developers, at different times and/or through different transformation techniques. However, as the number of transgenic loci increases over time, increasingly larger populations are needed to find the rare individual with the desired assortment of transgenic loci along with the non-transgenic elite traits. Introducing a transgene directly into a field cultivar would bypass the need to introgress the engineered trait. However, this necessitates separate transformations into numerous field cultivars, along with the characterization and regulatory approval of each independent transformation event. Reducing the number of segregating transgenic loci could be achieved if multiple traits are introduced at the same time, a preferred option if each of the many traits is new or requires re-engineering. If reengineering of previously introduced traits is not needed, then appending a new trait to an existing locus would be a rational strategy. The insertion of new DNA at a known locus can be accomplished by site- specific integration, through a host-dependent homology-based process, or a heterologous site-specific recombination system. Here, we discuss gene stacking through the use of site-specific recombinases.展开更多
为了解决骨形态发生蛋白10(bone morphogenetic protein 10,BMP10)前体蛋白proBMP10在中国仓鼠卵巢(CHO)细胞中由于Furin导致的表达产物不均一问题,构建了proBMP10在Furin酶切识别位点处的突变体proBMP10-1(R313K)和proBMP10-2(R316K),...为了解决骨形态发生蛋白10(bone morphogenetic protein 10,BMP10)前体蛋白proBMP10在中国仓鼠卵巢(CHO)细胞中由于Furin导致的表达产物不均一问题,构建了proBMP10在Furin酶切识别位点处的突变体proBMP10-1(R313K)和proBMP10-2(R316K),并分别将目的基因定点整合进CHO-K1-BAK-/BAX-基因组,成功构建了稳定表达目标蛋白质的重组CHO细胞株。结果表明,proBMP10-2 (R316K)不再被Furin切割,且具有生物活性,而proBMP10-1(R313K)仍然会被Furin切割。展开更多
Our lab has constructed a new nonviral vec-tor—hrDNA targeting vector(pHrneo). pHrneo is a human derived vector that can target gene into human ribosomal DNA(hrDNA) locus. In this study, we inserted expression casset...Our lab has constructed a new nonviral vec-tor—hrDNA targeting vector(pHrneo). pHrneo is a human derived vector that can target gene into human ribosomal DNA(hrDNA) locus. In this study, we inserted expression cassette of reconstructive hF Ⅷ (hFVIII-BDDAK39) to pHrneo to construct targeting vector: pHrneo-BDDAK39. Through electroporation of pHrneo-BDDAK39 into HT1080 cells, we identified the homologous recombinants by PCR and Southen blotting, and tested the expression of hFVIII- BDDAK39 in the hrDNA locus. The hFⅧ-BDDAK39 was successfully targeted into hrDNA locus of HT-1080 by pHrneo-BDDAK39, and the efficiency of site-specific inte-gration was 2.0×10?5. hFⅧ-BDDAK39 in hrDNA locus of HT-1080 is found to be able to express efficiently (32±5 ng·106 cells?1·24 h?1). Targeting vector pHrneo-BDDAK39 can find use in gene therapy for hemophilia.展开更多
Chinese hamster ovary(CHO)cells are widely used in biopharmaceuticals because of their high-density suspension culture,high safety,and high similarity between expressed exogenous proteins and natural proteins.However,...Chinese hamster ovary(CHO)cells are widely used in biopharmaceuticals because of their high-density suspension culture,high safety,and high similarity between expressed exogenous proteins and natural proteins.However,the level of exogenous protein expression decreases with increasing culture time;this phenomenon occurs due to the recombination of foreign genes into chromosomes through random integration.The present study integrated the foreign genes into a specific chromosomal site for stable expression based on CRISPR–Cas9 technology.The results showed that the exogenous proteins enhanced green fluorescent protein(EGFP)and human serum albumin(HSA)were successfully integrated into the vicinity of base 1969647 on chromosome NW_003613638.1 of CHO-K1 cells.The obtained positive monoclonal cell lines expressed all the corresponding exogenous proteins after 60 consecutive passages,and no significant differences in expression levels were observed.This study might provide a feasible method to construct a CHO cell line with long-term stable expression of exogenous proteins.展开更多
文摘The current method for combining transgenes into a genome is through the assortment of independent loci, a classical operating system compatible with transgenic traits created by different developers, at different times and/or through different transformation techniques. However, as the number of transgenic loci increases over time, increasingly larger populations are needed to find the rare individual with the desired assortment of transgenic loci along with the non-transgenic elite traits. Introducing a transgene directly into a field cultivar would bypass the need to introgress the engineered trait. However, this necessitates separate transformations into numerous field cultivars, along with the characterization and regulatory approval of each independent transformation event. Reducing the number of segregating transgenic loci could be achieved if multiple traits are introduced at the same time, a preferred option if each of the many traits is new or requires re-engineering. If reengineering of previously introduced traits is not needed, then appending a new trait to an existing locus would be a rational strategy. The insertion of new DNA at a known locus can be accomplished by site- specific integration, through a host-dependent homology-based process, or a heterologous site-specific recombination system. Here, we discuss gene stacking through the use of site-specific recombinases.
文摘为了解决骨形态发生蛋白10(bone morphogenetic protein 10,BMP10)前体蛋白proBMP10在中国仓鼠卵巢(CHO)细胞中由于Furin导致的表达产物不均一问题,构建了proBMP10在Furin酶切识别位点处的突变体proBMP10-1(R313K)和proBMP10-2(R316K),并分别将目的基因定点整合进CHO-K1-BAK-/BAX-基因组,成功构建了稳定表达目标蛋白质的重组CHO细胞株。结果表明,proBMP10-2 (R316K)不再被Furin切割,且具有生物活性,而proBMP10-1(R313K)仍然会被Furin切割。
基金This work was supported by Chinese 973 Projects (Grant No. 2004CB518800)； 863 Projects (Grant Nos. 2002BA7IIA07-08, 2002BA7l1A07-03 & 2002AA227011)；the National Natural Science Foundation of China (Grant No. 31830200) ； Life Science Research Foundation of Hunan Province.
文摘Our lab has constructed a new nonviral vec-tor—hrDNA targeting vector(pHrneo). pHrneo is a human derived vector that can target gene into human ribosomal DNA(hrDNA) locus. In this study, we inserted expression cassette of reconstructive hF Ⅷ (hFVIII-BDDAK39) to pHrneo to construct targeting vector: pHrneo-BDDAK39. Through electroporation of pHrneo-BDDAK39 into HT1080 cells, we identified the homologous recombinants by PCR and Southen blotting, and tested the expression of hFVIII- BDDAK39 in the hrDNA locus. The hFⅧ-BDDAK39 was successfully targeted into hrDNA locus of HT-1080 by pHrneo-BDDAK39, and the efficiency of site-specific inte-gration was 2.0×10?5. hFⅧ-BDDAK39 in hrDNA locus of HT-1080 is found to be able to express efficiently (32±5 ng·106 cells?1·24 h?1). Targeting vector pHrneo-BDDAK39 can find use in gene therapy for hemophilia.
文摘Chinese hamster ovary(CHO)cells are widely used in biopharmaceuticals because of their high-density suspension culture,high safety,and high similarity between expressed exogenous proteins and natural proteins.However,the level of exogenous protein expression decreases with increasing culture time;this phenomenon occurs due to the recombination of foreign genes into chromosomes through random integration.The present study integrated the foreign genes into a specific chromosomal site for stable expression based on CRISPR–Cas9 technology.The results showed that the exogenous proteins enhanced green fluorescent protein(EGFP)and human serum albumin(HSA)were successfully integrated into the vicinity of base 1969647 on chromosome NW_003613638.1 of CHO-K1 cells.The obtained positive monoclonal cell lines expressed all the corresponding exogenous proteins after 60 consecutive passages,and no significant differences in expression levels were observed.This study might provide a feasible method to construct a CHO cell line with long-term stable expression of exogenous proteins.
