Generation and characterization of transgenic mice with the full-length human DMD gene.
We report the generation of mice with an intact and functional copy of the 2.3-megabase human dystrophin gene (hDMD), the largest functional stretch of human DNA thus far integrated into a mouse chromosome. Yeast spheroplasts containing an artificial chromosome with the full-length hDMD gene were fused with mouse embryonic stem cells and were subsequently injected into mouse blastocysts to produce transgenic hDMD mice. Human-specific PCR, Southern blotting, and fluorescent in situ hybridization techniques demonstrated the intactness and stable chromosomal integration of the hDMD gene on mouse chromosome 5. Expression of the transgene was confirmed by RT-PCR and Western blotting. The tissue-specific expression pattern of the different DMD transcripts was maintained. However, the human Dp427p and Dp427m transcripts were expressed at 2-fold higher levels and human Dp427c and Dp260 transcripts were expressed at 2- and 4-fold lower levels than their endogenous counterparts. Ultimate functional proof of the hDMD transgene was obtained by crossing of hDMD mice with dystrophin-deficient mdx mice and dystrophin and utrophin-deficient mdx x Utrn-/- mice. The hDMD transgene rescued the lethal dystrophic phenotype of the mdx x Utrn-/- mice. All signs of muscular dystrophy disappeared in the rescued mice, as demonstrated by histological staining of muscle sections and gene expression profiling experiments. Currently, hDMD mice are extensively used for preclinical testing of sequence-specific therapeutics for the treatment of Duchenne muscular dystrophy. In addition, the hDMD mouse can be used to study the influence of the genomic context on deletion and recombination frequencies, genome stability, and gene expression regulation.
't Hoen
Peter A C
PA
Center for Human and Clinical Genetics, Leiden University Medical Center, Postal Zone S4-P, PO Box 9600, 2300 RC Leiden, The Netherlands. p.a.c.hoen@lumc.nl
de Meijer
Emile J
EJ
Boer
Judith M
JM
Vossen
Rolf H A M
RH
Turk
Rolf
R
Maatman
Ronald G H J
RG
Davies
Kay E
KE
van Ommen
Gert-Jan B
GJ
van Deutekom
Judith C T
JC
den Dunnen
Johan T
JT
D000818:Animals; D001755:Blastocyst / Q000166:cytology / Q000378:metabolism; D002875:Chromosomes / Q000235:genetics / Q000378:metabolism; D003433:Crosses, Genetic; D004353:Drug Evaluation, Preclinical; D016189:Dystrophin / Q000096:biosynthesis* / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics*; D018014:Gene Transfer Techniques; D042822:Genomic Instability / Q000235:genetics; D006801:Humans; D051379:Mice; D018101:Mice, Inbred mdx / Q000235:genetics / Q000378:metabolism; D008822:Mice, Transgenic / Q000235:genetics / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism*; D020388:Muscular Dystrophy, Duchenne / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D009928:Organ Specificity / Q000235:genetics; D049411:Utrophin / Q000235:genetics / Q000378:metabolism
C484258:DMD protein, human; D016189:Dystrophin; D049411:Utrophin