Genetic manipulation of murine embryonic stem cells with enhanced green fluorescence protein and sulfatase-modifying factor I genes.
OBJECTIVE
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-1alpha (hEF-1alpha) 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-1alpha promoter resulted in more homogeneous eGFP expression, with approximately 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-1alpha is a stronger promoter than CMV for ES cells and over-expression of eGFP may affect cell growth and contribute to unstable gene expression.
Zhao
Guoying
G
Lysosomal Diseases Research Unit, SA Pathology at the Women's and Children's Hospital, North Adelaide, Australia. holly_zhao@hotmail.com
Karageorgos
Litsa
L
Hutchinson
Rhonda G
RG
Hopwood
John J
JJ
Hemsley
Kim
K
D000818:Animals; D053595:Embryonic Stem Cells / Q000166:cytology / Q000502:physiology*; D015316:Genetic Therapy; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D009084:Mucopolysaccharidosis III / Q000235:genetics / Q000378:metabolism / Q000628:therapy; D050862:Oxidoreductases Acting on Sulfur Group Donors; D020648:Peptide Elongation Factor 1 / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D013429:Sulfatases / Q000235:genetics* / Q000378:metabolism; D019076:Transgenes
D020648:Peptide Elongation Factor 1; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D050862:Oxidoreductases Acting on Sulfur Group Donors; C519507:Sumf1 protein, mouse; D013429:Sulfatases