Quantification of normal dystrophin mRNA following myoblast transplantation in mdx mice.
A mutagenesis RT-PCR method was used to detect normal dystrophin mRNA following the injection of normal myoblasts in mdx mice using two immunosuppressors. A specific sequence of the dystrophin mRNA (257 bp) was amplified by RT-PCR from the muscle total RNA. MaeIII digestion of the amplified products allowed us to distinguish the normal messenger of dystrophin from the dystrophic one and to establish the percentage of normal and of dystrophic (mdx) dystrophin mRNA. Normal dystrophin mRNA was detected using this technique in mdx muscles transplanted with histocompatible normal myoblasts. For this type of transplantation, no significant difference in the percentage of normal dystrophin mRNA was observed between immunosuppressed mice and those not immunosuppressed. No normal dystrophin mRNA was, however, observed in mdx mice following histoincompatible normal myoblast transplantation without immunosuppression. When such transplantations were done in mice immunosuppressed with cyclosporine or FK-506, normal dystrophin mRNA accounted for 31% and 36% of the total dystrophin mRNA, respectively. In fact, one animal immunosuppressed with FK-506 expressed as much as 57% of normal dystrophin mRNA. These results thus show that FK-506 makes it possible to restore dystrophin expression to a level comparable to that observed in DMD carriers that are usually asymptomatic.
Asselin
I
I
Université Laval, Département d'Anatomie, PQ, Canada.
Tremblay
M
M
Vilquin
J T
JT
Guérette
B
B
Roy
R
R
Tremblay
J P
JP
D000818:Animals; D000906:Antibodies / Q000276:immunology; D001483:Base Sequence; D017690:Cell Transplantation; D002478:Cells, Cultured; D015342:DNA Probes; D016189:Dystrophin / Q000096:biosynthesis*; D005434:Flow Cytometry; D007165:Immunosuppression Therapy; D051379:Mice; D008810:Mice, Inbred C57BL; D018101:Mice, Inbred mdx; D008969:Molecular Sequence Data; D009132:Muscles / Q000166:cytology / Q000196:embryology* / Q000378:metabolism; D009137:Muscular Dystrophy, Animal / Q000378:metabolism*; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D012508:Sarcolemma / Q000378:metabolism; D033581:Stem Cell Transplantation
D000906:Antibodies; D015342:DNA Probes; D016189:Dystrophin; D012333:RNA, Messenger