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A constitutively active Gαi3 protein corrects the abnormal retinal pigment epithelium phenotype of Oa1-/- mice
Alejandra Young1, Ying Wang, Novruz B Ahmedli
1Jules Stein Eye Institute, University of California Los Angeles, Los Angeles, California, United States of America ; Molecular Biology Institute, University of California Los Angeles, Los Angeles, California, United States of America.
Purpose:
Ocular Albinism type 1 (OA1) is a disease caused by mutations in the OA1 gene and characterized by the presence of macromelanosomes in the retinal pigment epithelium (RPE) as well as abnormal crossing of the optic axons at the optic chiasm. We showed in our previous studies in mice that Oa1 activates specifically Gαi3 in its signaling pathway and thus, hypothesized that a constitutively active Gαi3 in the RPE of Oa1-/- mice might keep on the Oa1 signaling cascade and prevent the formation of macromelanosomes. To test this hypothesis, we have generated transgenic mice that carry the constitutively active Gαi3 (Q204L) protein in the RPE of Oa1-/- mice and are now reporting the effects that the transgene produced on the Oa1-/- RPE phenotype.
Methods:
Transgenic mice carrying RPE-specific expression of the constitutively active Gαi3 (Q204L) were generated by injecting fertilized eggs of Oa1-/- females with a lentivirus containing the Gαi3 (Q204L) cDNA. PCR, Southern blots, Western blots and confocal microscopy were used to confirm the presence of the transgene in the RPE of positive transgenic mice. Morphometrical analyses were performed using electron microscopy to compare the size and number of melanosomes per RPE area in putative Oa1-/-, Gαi3 (Q204L) transgenic mice with those of wild-type NCrl and Oa1-/- mice.
Results:
We found a correlation between the presence of the constitutively active Gαi3 (Q204L) transgene and the rescue of the normal phenotype of RPE melanosomes in Oa1-/-, Gαi3 (Q204L) mice. These mice have higher density of melanosomes per RPE area and a larger number of small melanosomes than Oa1-/- mice, and their RPE phenotype is similar to that of wild-type mice.
Conclusions:
Our results show that a constitutively active Gαi3 protein can by-pass the lack of Oa1 protein in Oa1-/- mice and consequently rescue the RPE melanosomal phenotype.
