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  • Title: [Features of transfection and expression of cDNA for the human dystrophin gene, delivered into muscles of mdx mice using MF-2 synthetic microspheres].
    Author: Baranov AN, Kiselev AV, Ivashchenko TE, Kashcheeva TK, Baranov VS.
    Journal: Genetika; 1999 Jan; 35(1):22-7. PubMed ID: 10330608.
    Abstract:
    The number of dystrophin-positive myofibers (DPM), that appeared in different skeletal muscles of mdx mice after a single injection of synthetic microspheres containing the full-length human dystrophin cDNA within the pHSADy expressing vector into femoral quadriceps muscle, was examined on cryostat sections. Injection of 25 micrograms cDNA resulted in the occurrence of 1, 2.4, 5.8 and 4.8% of DPM in the treated muscle in 1, 7, 21, and 60 days after the injection respectively. 7, 21, and 60 days after the treatment, these values comprised 2.1, 4.3 and 1% in the same muscle of the contralateral leg, and 5.5, 8.4, and 1% in the gluteal muscle. Expression of the full-length human dystrophin (427 kDa) in the muscle of the transfected mdx mice was observed. The presence of the transfected construction in skeletal muscles, heart, brain, lungs, and fetuses was demonstrated PCR. Utilization of the FISH technique with biotinilated pHSADy construct as a DNA probe showed that 7 days after the injection, the MF-2 microspheres were present in 70% of myoblast nuclei, in 64% of nuclei of gluteal muscles, and in 62% of the contralateral quadriceps nuclei. 21 days after the treatment, these values were 41, 29, and 45%, respectively. The MF-2 microsphere were detected in the nuclei of the blood, brain, heart, and lung cells, as well as in the placenta and tissues of 18-day-old fetuses. Our results demonstrated the high efficiency of microsphere-mediated transfer of gene constructs into cell nuclei, their long-term intranuclear persistence, and the ability to direct expression for at least 2 months after injection. The MF-2 microspheres attract special interest in respect to the targeted delivery of gene constructs into the nuclei.
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