March 2012
Volume 53, Issue 14
ARVO Annual Meeting Abstract  |   March 2012
Progeny Of Pronuclear Injections Of Mutant Human Mitochondrial Genes
Author Affiliations & Notes
  • Hong Yu
    Ophthalmology, Bascom Palmer Eye Inst, Univ of Miami, Miami, Florida
  • Tsung-Han Chou
    Ophthalmology, Bascom Palmer Eye Inst, Univ of Miami, Miami, Florida
  • Vittorio Porciatti
    Bascom Palmer Eye Inst, Univ of Miami Miller Sch Med, Miami, Florida
  • William W. Hauswirth
    Ophthalmology, University of Florida, Gainesville, Florida
  • Vince Chiodo
    Ophthalmology, University of Florida, Gainesville, Florida
  • Sanford L. Boye
    Ophthalmology, University of Florida, Gainesville, Florida
  • John Guy
    Ophthalmology, Bascom Palmer Eye Inst, Univ of Miami, Miami, Florida
  • Footnotes
    Commercial Relationships  Hong Yu, None; Tsung-Han Chou, None; Vittorio Porciatti, None; William W. Hauswirth, None; Vince Chiodo, None; Sanford L. Boye, None; John Guy, None
  • Footnotes
    Support  R01 EY017141
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 5581. doi:
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    • Get Citation

      Hong Yu, Tsung-Han Chou, Vittorio Porciatti, William W. Hauswirth, Vince Chiodo, Sanford L. Boye, John Guy; Progeny Of Pronuclear Injections Of Mutant Human Mitochondrial Genes. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5581.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : To generate a transgenic mouse with human mutated G11778A ND4 mtDNA.

Methods: : The mutant human ND4 gene with a FLAG epitope tag followed by mitochondrial encoded mCherry under control of the mitochondrial promoter (sc-HSP-ND4G1019AmtmCherry) was packaged with mito-targeted (COX8) scAAV that was injected into fertilized oocytes using a standard pronuclear microinjection protocol. Briefly, hybrid (C57BL/6J x DBA/2J) F1 mice, referred to as B6D2F1, were obtained for production of fertilized oocytes. Superovulation was stimulated by intraperitoneal injection of B6D2F1 females with 5 IU pregnant mare serum gonadotropin followed 46-48 h later by injection of 5 IU human chorionic gonadotropin. Following HCG injection, females were mated with B6D2F1 stud males and oocytes harvested the following day. A concentrated AAV virus stock (1.06E+12 VG/ml) was microinjected into the pronuclei of fertilized oocytes using a continuous flow injection mode. Surviving eggs were implanted into the ampulla of pseudopregnant females. After weaning the resulting offspring were analyzed for the presence of human ND4 in isolated mouse mitochondria was assessed by PCR, sequencing and immunohistochemistry. Visual function was monitored by pattern electroretinography (PERG).

Results: : PCR amplification of mitochondrial DNA, isolated from different organs of transgenic mice and their progeny including retina, optic nerve, brain, liver, heart and muscle, revealed expected 500bp bands. The corresponding DNA sequences confirmed that PCR products are human ND4 and mCherry. Fluorescence of mCherry could be visualized in retina and optic nerve head of live mice by using a laser scanning ophthalmoscope. Longitudinal retina sections revealed ND4FLAG in RGC layer and co-localization with the mitochondrial membrane protein VDAC porin. Pattern electroretinogram (PERG) showed that the amplitudes decreased dramatically with aging (p=0.006) in transgenice mice and transgenic mice had significantly lower PERG amplitude compared to normal mice of the same strain (p=0.02).

Conclusions: : Despite multiorgan dissemination of mutant G11778A ND4, our transgenic mice developed only visual loss, as also seen in LHON patients. Pronuclear injection of mutant human mitochondrial genes is an innovative biotechnical advance for evaluating LHON and likely other devastating neurodegenerative disorders with mitochondrial etiology and their treatment.

Keywords: gene transfer/gene therapy • retinal degenerations: hereditary • transgenics/knock-outs 

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