Site-specific gene modification by oligodeoxynucleotides in mouse bone marrow-derived mesenchymal stem cells.
Synthetic oligodeoxynucleotides (ODNs) had been employed in gene modification and represent an alternative approach to 'cure' genetic disorders caused by mutations. To test the ability of ODN-mediated gene repair in bone marrow-derived mesenchymal stem cells (MSCs), we established MSCs cell lines with stably integrated mutant neomycin resistance and enhanced green fluorescent protein reporter genes. The established cultures showed morphologically homogenous population with phenotypic and functional features of mesenchymal progenitors. Transfection with gene-specific ODNs successfully repaired targeted cells resulting in the expression of functional proteins at relatively high frequency approaching 0.2%. Direct DNA sequencing confirmed that phenotype change resulted from the designated nucleotide correction at the target site. The position of the mismatch-forming nucleotide was shown to be important structural feature for ODN repair activity. The genetically corrected MSCs were healthy and maintained an undifferentiated state. Furthermore, the genetically modified MSCs were able to engraft into many tissues of unconditioned transgenic mice making them an attractive therapeutic tool in a wide range of clinical applications.
Flagler
K
K
Department of Dermatology and Cutaneous Biology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Alexeev
V
V
Pierce
E A
EA
Igoucheva
O
O
D000818:Animals; D001483:Base Sequence; D018929:Cell Culture Techniques; D004277:DNA, Single-Stranded / Q000008:administration & dosage*; D004352:Drug Resistance, Microbial / Q000235:genetics*; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics; D045164:Mesenchymal Stem Cell Transplantation; D059630:Mesenchymal Stem Cells / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D052416:Targeted Gene Repair; D014162:Transfection / Q000379:methods
D004277:DNA, Single-Stranded; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins