Genetic manipulation of murine embryonic stem cells with enhanced green fluorescence protein and sulfatase-modifying factor I genes

Guoying Zhao, Litsa Karageorgos, Rhonda G. Hutchinson, John J. Hopwood, Kim Hemsley

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Background aims. Mucopolysaccharidosis 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. Methods. In 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. Results. During 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. Conclusions. Our 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.

Original languageEnglish
Pages (from-to)400-407
Number of pages8
JournalCytotherapy
Volume12
Issue number3
DOIs
Publication statusPublished - 2010
Externally publishedYes

Keywords

  • Embryonic stem cells
  • Enhanced green fluorescence protein
  • Gene expression
  • Mucopolysaccharidosis type IIIA
  • Sulfamidase
  • Sulfatase-modifying factor I

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