Application of in vitro Myo-differentiation of non-muscle cells to enhance gene expression and facilitate analysis of muscle proteins.
Introduction of the myogenic-determination gene MyoD forces non-muscle cell cultures into myogenesis, thereby inducing expression of muscle-specific proteins and facilitating their analysis. In several MyoD-transfected fibroblasts, immunohistochemical detection showed expression of desmin after three days, of titin after five days and of dystrophin after seven days. Cell fusion (myotube formation) could be observed after five days. After nine days a fraction of the cells showed a striated titin pattern, indicating an advanced state of muscle differentiation. Dystrophin (the protein absent in Duchenne Muscular Dystrophy patients) can be detected in MyoD-transfected and differentiated fibroblasts from healthy individuals, and is absent in those of patients. MyoD-transfection increases transcription of the dystrophin gene, facilitating RNA-based mutation detection. Using RNA from MyoD-transfected, differentiated fibroblasts of a deceased patient with an unknown, non-deletion mutation, we were able to identify a CGA-->TGA nonsense mutation in the rod domain at basepair 6492 and to establish a rapid mutation specific test for future diagnosis of the mutation in his relatives.
Roest
P A
PA
MGC-Department of Human Genetics, Leiden University, The Netherlands.
van der Tuijn
A C
AC
Ginjaar
H B
HB
Hoeben
R C
RC
Hoger-Vorst
F B
FB
Bakker
E
E
den Dunnen
J T
JT
van Ommen
G J
GJ
D001482:Base Composition; D001483:Base Sequence; D002454:Cell Differentiation; D002459:Cell Fusion; D002478:Cells, Cultured; D017931:DNA Primers; D003893:Desmin / Q000096:biosynthesis; D016189:Dystrophin / Q000096:biosynthesis / Q000235:genetics; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism / Q000473:pathology; D015870:Gene Expression; D006801:Humans; D008297:Male; D009124:Muscle Proteins / Q000032:analysis / Q000096:biosynthesis*; D018482:Muscle, Skeletal / Q000166:cytology*; D009136:Muscular Dystrophies / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D017570:MyoD Protein / Q000032:analysis / Q000096:biosynthesis*; D009928:Organ Specificity; D010375:Pedigree; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012016:Reference Values; D012867:Skin / Q000166:cytology* / Q000378:metabolism / Q000473:pathology; D014162:Transfection / Q000379:methods
D017931:DNA Primers; D003893:Desmin; D016189:Dystrophin; D009124:Muscle Proteins; D017570:MyoD Protein