研究人员发明一种新工具,它在促成多能干细胞中基因的选择性表达时,不会影响已经分化的细胞,这一新成果发表在日前在线出版的《自然—方法学》期刊上。
诱导多能干细胞(iPS)是通过程序化重组成熟细胞而制成的,它们有分化成各种成熟细胞类型的潜能,但程序化重组过程的效率非常低。
James Ellis和同事合作,讲述了一种可促成基因在iPS细胞中独自表达的病毒载体如何提高了iPS细胞的选择性。通过一种抗生素耐药性基因的表达,它们用这些运输器将抗生素耐药性告知给新出现的iPS细胞。Ellis和同事从雷特氏综合征患者和模型小鼠体内取出人类和小鼠的iPS细胞。雷特氏综合征是一种神经失调疾病。新方法将被证明可应用于实验室里的疾病研究,因为它能简单地分离出小鼠和人类的iPS细胞系。
新方法很可能应用于制作疾病的试管模型,而iPS细胞则是通过重组取自特定疾病患者的细胞衍生。(生物谷Bioon.com)
生物谷推荐原始出处:
Nature Methods 6, 370 - 376 (2009) 26 April 2009 | doi:10.1038/nmeth.1325
Isolation of human iPS cells using EOS lentiviral vectors to select for pluripotency
Akitsu Hotta1,2, Aaron Y L Cheung1,3, Natalie Farra1,3, Kausalia Vijayaragavan4,6, Cheryle A Séguin1,2,6, Jonathan S Draper1,6, Peter Pasceri1, Irina A Maksakova5, Dixie L Mager5, Janet Rossant1,2,3, Mickie Bhatia4 & James Ellis1,2,3
Induced pluripotent stem (iPS) cells may be of use in regenerative medicine. However, the low efficiency of reprogramming is a major impediment to the generation of patient-specific iPS cell lines. Here we report the first selection system for the isolation of human iPS cells. We developed the EOS (Early Transposon promoter and Oct-4 (Pou5f1) and Sox2 enhancers) lentiviral vector to specifically express in mouse and human embryonic stem cells but not in primary fibroblasts. The bicistronic EOS vector marked emerging mouse and human iPS cell colonies with EGFP, and we used puromycin selection to aid the isolation of iPS cell lines that expressed endogenous pluripotency markers. These lines differentiated into cell types from all three germ layers. Reporter expression was extinguished upon differentiation and therefore monitored the residual pluripotent cells that form teratomas. Finally, we used EOS selection to establish Rett syndrome–specific mouse and human iPS cell lines with known mutations in MECP2.
1 Developmental and Stem Cell Biology Program, Toronto, Ontario, Canada.
2 Ontario Human iPS Cell Facility, SickKids, Toronto, Ontario, Canada.
3 Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
4 McMaster Stem Cell and Cancer Research Institute, Michael G. DeGroote School of Medicine, and Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada.
5 Terry Fox Laboratory, British Columbia Cancer Agency, and Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada.
6 Present addresses: Department of Regenerative Cardiology, Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain (K.V.), 7 Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, The University of Western Ontario, London, Ontario, Canada (C.A.S.) and McMaster Stem Cell and Cancer Research Institute, Michael G. DeGroote Centre for Learning and Discovery, Hamilton, Ontario, Canada (J.S.D.).