Purchase this article with an account.
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.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
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.
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.
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.
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.
This PDF is available to Subscribers Only