Electroretinography and Retinal Pathology of Two Lines of ELOVL4 270stmt Transgenic Pigs. Transgene Expression Levels Correlate With the Degree of Stargardt-Like Macular Degeneration

Abstract

Purpose:: Stargardt-like macular dystrophy 3 (STGD3) is a progressive juvenile onset macular disease. We have produced transgenic pigs as a model for this disease with the autosomal dominant ELOVL4 270stmt mutation. The mutant ELOVL4 is expressed as a fusion protein with EYFP for ease of detection. We examined the pathology of two transgenic lines, one of which was known to have higher transgene expression than the other; lines 913 and 304. We used both electroretinography (ERG) and gross morphologic analysis to examine the pathology in the two lines.

Methods:: Electroretinography was performed on anesthetized transgenic pigs. All animals were dark adapted for 15 minutes prior to ERG. Histology was performed on frozen sections of 4% paraformaldehyde fixed samples. Direct fluorescent examination of the EYFP ELOVL4 270stmt fusion protein was performed using either epifluorescent microscopy or confocal laser microscopy. Propidium iodide was used for nuclear staining on some of the samples. Hemotoxin and eosin staining was also performed.

Results:: We have previously determined different levels of expression between the transgenic lines 913 and 304 by direct fluorescent microscopy of live retinal tissue. Line 913 is a low expressing line. ERG analysis of the founder transgenic pig at 2 yrs of age as well as two 1 yr progeny from this line indicated a slower progression of pathology than seen in the high expressing line 304. At 2 yrs the 913 founder animal had a diminished ERG (B-scot 79.9 uV and B-phot 49.6 uV). At one yr of age, the 913 line pigs have normal ERG response. The high expressing line, 304, at 1.75 yrs had a greatly diminished ERG (B-scot 43.6 uV and B-phot 19.7 uV). Consistent with STGD3 pathology, EYFP ELOVL4 270stmt expression was found in the ISL and ONL. Furthermore, we found that the nuclear number of the outer nuclear layer in line 304 is less than the 913 line or wild type pig. The ONL at the area centralis is 5-6 layers thick for a wild type pig, 4 layers for the 304 animal, and 5 layers for the line 913 transgenic pigs.

Conclusions:: Two lines of ELOVL4 270stmt transgenic pigs with different levels of expression show indications of pathology consistent with STGD3. The progression of the disease is faster with the high expressing 304 line.

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