Fluorescent protein tdTomato is deleterious to the retina

Abstract

Purpose : Fluorescent proteins (FPs) are widely used in imaging techniques to investigate cellular molecular interactions and trace biological events. However, some FPs have been demonstrated to cause undesirable cellular damage. So far, the effects of red fluorescent protein (RFP) tandem dimer Tomato (tdTomato) on retina remains unknown. Here we study the effects of tdTomato expression on mice retina.

Methods : Electroretinography (ERG) was carried out to investigate the changes of retinal function comparing tdTomato mice with C57BL/6J wild-type control mice. Optokinetic Response (OKR) was measured to detect visual contrast sensitivity. Color fundus camera and high-resolution spectral-domain optical coherence tomo-graphy (SD-OCT) were taken to compare the retinal structural alteration. Immunofluorescence staining was used to label proteins in retina. Transmission Electron Microscope (TEM) was used to observe the retinal ultrastructure. Expression levels of mitochondrial specific genes were quantified via quantitative real-time PCR (qPCR).

Results : The tdTomato mice showed progressively attenuated electroretinogram (ERG) responses in scotopic a-wave and photopic b-wave, and reduction of visual contrast sensitivity was detected via OKR. Mitochondrial vacuolation and broken mitochondrial crest were found by TEM. And down-regulated mitochondrial DNA (mtDNA) expression were found in tdTomato mice. The apparent changes in retina of color fundus camera, SD-OCT and immunofluorescence staining measurements were not found.

Conclusions : tdTomato expression cause functional and ultrastructural defects in the retinas of tdTomato mice, indicates potential toxicity of tdTomato to mouse retina. Mitochondria should be a potential target of the fluorescent protein tdTomato that hazard to the mouse retina. Our findings provide valuable insights into using the tdTomato mice more efficiently and reasonably.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

 

Figure 2. tdTomato decreased the Electroretinogram (ERG) responses and Visual sensitivity of mice. Quantitative measurements of scotopic and photopic, representative scotopic of P1m (A), P3m (B), P6m(C) and P9m(D) mice. Quantitative measurements of scotopic and photopic. ERG recordings were stimulated at different intensities, and obtained from C57BL/6J mice and tdTomato mice at P1m (A), P3m (B), P6m(C) and P9m(D). C57BL/6J mice: n=8, 8, 8 and 10 for P1m, P3m, P6m and P9m respectively; tdTomato mice: n=8, 7, 6 and 9 for P1m, P3m, P6m and P9m respectively. (E) Schematic of Optokinetic set-up (see materials and methods). The mice were detected contrast sensitivity at 120 cycles. (F, G) Mice were measured optokinetic responses at P6m (F) and P9m (G). C57BL/6J mice: n=13 in P6m and 10 in P9m; tdTomato mice: n=14 in P6m and 12 in P9m. Error bars represent as mean ±SEM. n.s.: no statistical significance, *P < 0.05, **P < 0.01, Student t-test.

 

Figure 4. Retinal ultrastructure indicated the mitochondrial abnormalities in tdTomato mice at P3m. (A) Normal mitochondria of C57BL/6J mice under electron microscope. (B and C) Retinal ultrastructure of tdTomato mice indicated the mitochondrial vacuolation (purple asterisks in B) and the mitochondrial crest broke (red asterisks in C). The magnification of electron microscope is 20,000 folds.