Purpose:To construct a novel AAV-mediated gene delivery of the human ND4 complex I subunit and to detect its expression level in mitochondria for potential application in gene therapy for Leber's hereditary optic ...Purpose:To construct a novel AAV-mediated gene delivery of the human ND4 complex I subunit and to detect its expression level in mitochondria for potential application in gene therapy for Leber's hereditary optic neuropathy (LHON). Methods:A novel type of normal human ND4 gene was synthesized artificially to contain a mitochondrial targeting sequence that induces the translocation of this gene into mitochondria.This recombinant adeno-associated virus type 2/serotype 2 (rAAV2/2)-mediated NADH dehydrogenase subunit 4 (ND4) gene was constructed, purified, condensed, and amplified by PCR.The physical titer of rAAV2/2-ND4 was determined by slot-blot hybridization using a digoxigeninlabeled H1 probe.Expression of ND4 in mitochondria was evaluated by immunofluorescence. Results: The constructed rAAV2/2-ND4 specifically amplified the target gene band of ND4 and the physical titer of ND4 gene was 1.0×1011 vg/mL,confirming that the recombinant adenovirus vector contained the ND4 target gene. Expression of the ND4 gene was detected in mitochondria by immunofluorescence. Conclusion:A new type of rAAV2/2-ND4 was successfully constructed and may have potential in gene therapy for LHON.展开更多
基金National Natural Science Foundation of China(30640048)the Science Foundation of the Key Laboratory of Biotic Environment andEcological Safetyin Anhui Province, China
基金financially supported by National Natural Science Fund.Molecule mechanism of the resistance of HRD1 against diabetic retinopathy (NO.81271015)Molecule mechanism of the resistance of P58IPK against diabetic retinopathy (NO.30872823)
文摘Purpose:To construct a novel AAV-mediated gene delivery of the human ND4 complex I subunit and to detect its expression level in mitochondria for potential application in gene therapy for Leber's hereditary optic neuropathy (LHON). Methods:A novel type of normal human ND4 gene was synthesized artificially to contain a mitochondrial targeting sequence that induces the translocation of this gene into mitochondria.This recombinant adeno-associated virus type 2/serotype 2 (rAAV2/2)-mediated NADH dehydrogenase subunit 4 (ND4) gene was constructed, purified, condensed, and amplified by PCR.The physical titer of rAAV2/2-ND4 was determined by slot-blot hybridization using a digoxigeninlabeled H1 probe.Expression of ND4 in mitochondria was evaluated by immunofluorescence. Results: The constructed rAAV2/2-ND4 specifically amplified the target gene band of ND4 and the physical titer of ND4 gene was 1.0×1011 vg/mL,confirming that the recombinant adenovirus vector contained the ND4 target gene. Expression of the ND4 gene was detected in mitochondria by immunofluorescence. Conclusion:A new type of rAAV2/2-ND4 was successfully constructed and may have potential in gene therapy for LHON.
