TY - JOUR
T1 - Genetic manipulation of murine embryonic stem cells with enhanced green fluorescence protein and sulfatase-modifying factor I genes
AU - Zhao, Guoying
AU - Karageorgos, Litsa
AU - Hutchinson, Rhonda G.
AU - Hopwood, John J.
AU - Hemsley, Kim
PY - 2010
Y1 - 2010
N2 - 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.
AB - 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.
KW - Embryonic stem cells
KW - Enhanced green fluorescence protein
KW - Gene expression
KW - Mucopolysaccharidosis type IIIA
KW - Sulfamidase
KW - Sulfatase-modifying factor I
UR - http://www.scopus.com/inward/record.url?scp=77950550603&partnerID=8YFLogxK
U2 - 10.3109/14653241003695026
DO - 10.3109/14653241003695026
M3 - Article
AN - SCOPUS:77950550603
VL - 12
SP - 400
EP - 407
JO - Cytotherapy
JF - Cytotherapy
SN - 1465-3249
IS - 3
ER -