Genetic manipulation of murine embryonic stem cells with enhanced green fluorescence protein and sulfatase-modifying factor I genes |
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| Authors: | Guoying Zhao Litsa Karageorgos Rhonda G. Hutchinson John J. Hopwood Kim Hemsley |
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| Affiliation: | 1. Lysosomal Diseases Research Unit, SA Pathology at the Women''s and Children''s Hospital, North Adelaide, Australia;2. Department of Cytogenetics, SA Pathology at the Women''s and Children''s Hospital, North Adelaide, Australia;3. Department of Paediatrics, University of Adelaide, North Adelaide, Australia;1. Neurogenetic Clinic, Department of Neurology, Schneider Children''s Medical Center of Israel, Petah Tiqwa, Affiliated with Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel;2. Pediatric Neurology and Child Development Center, Hillel Yaffe Medical Center, Hadera, Affiliated with Rappaport Faculty of Medicine, Technion—Israel Institute of Technology, Haifa, Israel;3. Department of Pathology, Hadassah University Hospital, Hebrew University-Hadassah Medical School, Jerusalem, Israel;4. Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/Université de Strasbourg, et Collège de France, 67404 Illkirch, France;3. The Wistar Institute, Philadelphia, Pennsylvania 19104;4. Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104;5. Department of Molecular Genetics, Lerner Research Institute, Cleveland, Ohio 44295;1. Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts;2. Division of Plastic and Reconstructive Surgery, Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts;1. Department of Pediatric Hematology, Guangzhou Women and Children''s Medical Center, Guangzhou, China;2. Department of Pediatric Endocrinology and Genetic Metabolism, Guangzhou Women and Children''s Medical Center, Guangzhou, China;1. Department of Dermatology and Venereology, University of Cologne, Germany;2. Institute of Virology, University of Cologne, Germany;3. National Reference Center for Papillomaviruses and Polyomaviruses, Germany;4. CIO, Center for Integrated Oncology Cologne – Bonn, Cologne, Germany |
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| Abstract: | Background aimsMucopolysaccharidosis type IIIA (MPS IIIA) is a lysosomal storage disorder (LSD) in which an absence of sulfamidase results in incomplete degradation and subsequent accumulation of its substrate, heparan sulfate. Most neurodegenerative LSD remain untreatable. However, therapy options, such as gene, enzyme end cell therapy, are under investigation. Previously, we have constructed an embryonic stem (ES) cell line (NS21) that over-expresses human sulphamidase as a potential treatment for murine MPS IIIA.MethodsIn the present study the sulfatase-modifying factor I (SUMF1) and enhanced green fluorescence protein (eGFP) genes were co-introduced under a cytomegalovirus (CMV) promoter into NS21 cells, to enhance further sulfamidase activity and provide a marker for in vivo cell tracking, respectively. eGFP was also introduced under the control of the human elongation factor-1α (hEF-1α) promoter to compare the stability of transgene expression.ResultsDuring differentiation of ES cells into glial precursors, SUMF1 was down-regulated and was hardly detectable by day 18 of differentiation. Likewise, eGFP expression was heterogeneous and highly unstable. Use of a human EF-1α promoter resulted in more homogeneous eGFP expression, with ～ 50% of cells eGFP positive following differentiation into glial precursors. Compared with NS21 cells, the outgrowth of eGFP-expressing cells was not as confluent when differentiated into glial precursors.ConclusionsOur data suggest that SUMF1 enhances sulfamidase activity in ES cells, hEF-1α is a stronger promoter than CMV for ES cells and over-expression of eGFP may affect cell growth and contribute to unstable gene expression. |
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