June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Generation of a transient (non-germline transmission) Mgp conditional-Knockout (c-KO) in the trabecular meshwork of living mice
Author Affiliations & Notes
  • Priyadarsini Asokan
    Ophthalmology, Univ of NC at Chapel Hill, Chapel Hill, North Carolina, United States
  • Laura Rodriguez Estevez
    Ophthalmology, Univ of NC at Chapel Hill, Chapel Hill, North Carolina, United States
  • Terete Borras
    Ophthalmology, Univ of NC at Chapel Hill, Chapel Hill, North Carolina, United States
    Gene Therapy Center, Univ of NC at Chapel Hill, Chapel Hill, North Carolina, United States
  • Footnotes
    Commercial Relationships   Priyadarsini Asokan, None; Laura Rodriguez Estevez, None; Terete Borras, None
  • Footnotes
    Support  EY011906; EY026220
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 3470. doi:
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      Priyadarsini Asokan, Laura Rodriguez Estevez, Terete Borras; Generation of a transient (non-germline transmission) Mgp conditional-Knockout (c-KO) in the trabecular meshwork of living mice. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3470.

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

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Abstract

Purpose : To establish and characterize the Mgp-floxed mouse for the study of eye stiffness in glaucoma. Previously, we reported the successful insertion of loxP sites flanking Mgp exons 2 and 3 in a mouse line using CRISPR/CAS9 technology. Here we seek to functionally characterize this floxed mouse by determining the positive recombination of the loxP sites in vitro and in vivo.

Methods : Primers were designed in the mouse genome at both sides of the loxP sites to amplify and easily discern the recombined allele (659 bp) from the non-recombined WT (2804 bp). Eyes were enucleated from 2-3 months old homozygous Mgp-loxP and WT mice. Primary angle cells were established from the TM region and cultured for 3 weeks. Whole anterior segments were dissected for stationary organ culture. An Adeno-GFP-2A-iCre virus encoding a fused messenger and separated GFP-Cre proteins was purchased, grown and purified in our laboratory. Cell and organ cultures from floxed and WT mice were infected with 9.4x1010 ifu Ad-GFP-2A-iCre and Ad-CMV-GFP as controls. Genomic DNA was extracted 48 h post-infection. In vivo, Mgp-loxP homozygous mice were injected IC in one eye with 3.8x109 ifu Ad-GFP-2A-iCre and contralateral control was uninjected. At 1 week, anterior segment tissues were processed for evaluation of TM GFP fluorescence. Genomic DNA was extracted from TM strips, amplified with designed primers and LongAmp Taq PCR kit.

Results : Fluorescence evaluation showed 80-90 % GFP transgene delivery. In vivo, a preferred TM delivery was confirmed by the presence of a whole mount green ring at the limbus area. Qualitative PCR gels showed a strong 659 bp fragment (recombined allele) on the DNA from all loxP cells/tissues infected with Ad-GFP-2A-iCre. The recombined fragment was not observed in control DNAs (Ad-GFP infected loxP and Ad-GFP-2A-iCre infected WT cells), which instead showed a 2804 fragment corresponding to the WT allele. No WT fragment was observed in loxP, Ad-GFP-2A-iCre infected cells DNA, indicating about 90% recombination.

Conclusions : The loxP sites of the Mgp-floxed mouse line are functional and can be recombined in vitro and in vivo by delivering the Cre protein. This model represents a unique opportunity to generate c-KOs by either gene transfer or by genetic crosses with promoter specific driven Cre mice. TM Mgp-cKO mice provide a unique tool for the study of stiffness in glaucoma.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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