کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
1929937 | 1050481 | 2011 | 6 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: A new chromosome 14-based human artificial chromosome (HAC) vector system for efficient transgene expression in human primary cells A new chromosome 14-based human artificial chromosome (HAC) vector system for efficient transgene expression in human primary cells](/preview/png/1929937.png)
The use of non-integrating human artificial chromosomes (HACs) in gene therapy possibly allows for safe and reliable genetic modification of human cells without insertional mutagenesis and/or unexpected oncogene activations. Although we previously demonstrated that the HAC provides long-term therapeutic erythropoietin (EPO) production in normal human primary fibroblasts (hPFs), the expression level of EPO was too low to provide medical benefits for human therapy. Thus, the next challenge for the application of this system in therapeutic purposes is to improve the transgene expression on HACs. Here, we newly constructed chromosome 14-based HACs and examined the effects of the telomere and promoter regions on the expression level of the tansgene in hPFs. We showed that the use of natural telomere/sub-telomere and enhancers within the 5′ untranslated region of the human ubiquitin C gene greatly increased (over 1000-fold) the EPO production in hPFs. Furthermore, we demonstrated the reprogramming of mouse embryonic fibroblasts by HAC-mediated introduction of four transcription factors, and established induced pluripotent stem cells with no trace of the HACs carrying multiple expression cassettes with large genome fragments. These results indicate that this HAC system could allow us to manipulate multiple transgenes efficiently in human primary cells, providing a promising tool not only for gene therapy but also for investigating genome functions in drug discoveries.
► We developed a new chromosome 14-based human artificial chromosome (HAC) vector system.
► The natural telomere and sub-telomere sequences on the HACs augmented the transgene expression.
► We demonstrated the presence of enhancers in the 5′ untranslated region of the ubiquitin C gene.
► This HAC system increased transgene expression by over 1000-fold compared with our previous data.
► We showed reprogramming of MEFs to induced pluripotent stem cells confirmed with no trace of the HAC.
Journal: Biochemical and Biophysical Research Communications - Volume 415, Issue 3, 25 November 2011, Pages 439–444