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Perkins. J Transl Genet Genom 2022;6:95-110 https://dx.doi.org/10.20517/jtgg.2021.47 Page 103

after the onset of transgene expression and most rods were missing by 5 dpf. Expression of the mCFP
caused rhodopsin mislocalization to the inner segments and rapid degeneration. It is possible that the
genomic integration sites of the Tg(XOPS:mCFP) transgene could have affected rod function, although this
is considered unlikely. Zebrafish carrying the Tg(XOPS:mCFP) transgene survived into adulthood but
lacked almost all rod photoreceptors. Cone photoreceptors survived into adulthood and cone morphology,
and the cone mosaic appeared normal in the transgenic animals. This was the first indication that rod
degeneration did not result in the secondary death of cones in zebrafish. The authors also noted an increase
in the proliferation of rod precursor cells within the ONL of Tg(XOPS:mCFP) adults. The Tg(XOPS:mCFP)
line has been instrumental in identifying the separate genetic pathways for rod and cone regeneration ,
[134]
[135]
identifying genes important for rod regeneration , and to demonstrate that an 11-cis-retinyl ester cycle is
[136]
critical to maintain cone vision .

Inducible rod death: Tg(rho:YFP-ntr)gmc500Tg, Tg(rho:Eco.NfsB-EGFP)nt19Tg, and Tg(rho:Eco.NfsB-
EGFP)nt20Tg
To study the effects of selective photoreceptor death in a controlled fashion, two groups independently
generated transgenic lines that show rod-specific expression of the E. coli nitroreductase enzyme fused to a
fluorescent reporter gene [137,138] . The nitroreductase enzyme (NTR, nfsB gene) reduces nitroimidazole
prodrugs, such as metronidazole (MTZ), into cytotoxic metabolites that crosslink DNA and cause rapid
death. When transgenic zebrafish are exposed to MTZ, any cells expressing NTR are selectively and
specifically eliminated. Montgomery et al. first published the Tg(rho:Eco.NfsB-eGFP)nt19Tg and
[138]
Tg(rho:Eco.NfsB-eGFP)nt20Tg alleles to investigate how rod death induces regeneration. Both lines utilize a
1.2 kb fragment of the zebrafish rhodopsin promoter to express a NTR-eGFP fusion protein specifically in
rods. In the absence of MTZ, rods express NTR-eGFP and GFP fluorescence is visible in the rod outer
segments and inner segments. Upon exposure to 10 mM MTZ for 24 h, all the rods in the retinas of the
Tg(rho:Eco.NfsB-eGFP)nt19Tg line rapidly degenerated within 48 h. Despite carrying the identical transgene,
the Tg(rho:Eco.NfsB-eGFP)nt20Tg transgenic line displayed NTR-eGFP expression in only a subset of rods.
The genomic integration sites of the transgene most certainly differed between the nt19Tg and nt20Tg alleles
and this may have influenced the expression patterns. When the Tg(rho:Eco.NfsB-eGFP)nt20Tg transgenic
fish were exposed to MTZ for 24 h, only those rods expressing NTR were destroyed and the non-expressing
rods survived. The difference in damage between the two lines allowed the authors to compare how the
retina responds to the loss of a subset of rods compared to the acute ablation of all rods photoreceptors.
When a subset of rods was destroyed in the Tg(rho:Eco.NfsB-eGFP)nt20Tg transgenic line, rod precursor
proliferation in the ONL was increased. The acute loss of all rods following MTZ treatment in
Tg(rho:Eco.NfsB-eGFP)nt19Tg transgenic animals triggered a regenerative response by Müller glia .
[138]
Independently, Ariga et al. and Walker et al. generated the Tg(rho:YFP-ntr)gmc500 transgenic line,
[139]
[140]
which utilizes a 3.7 kb fragment of the zebrafish rhodopsin promoter to drive a YFP-nfsB transgene. The
Tg(rho:YFP-ntr)gmc500 transgene was also expressed in all rod photoreceptors, indicating that the 3.7 kb
promoter and the 1.2 kb promoter both contain proper elements for rod-specific expression. Exposing
Tg(rho:YFP-ntr)gmc500 transgenic larvae to MTZ resulted in rod-specific death within 48 h. Time-lapse in
vivo imaging of demonstrated that peripheral macrophages and resident microglia rapidly transitioned to an
amoeboid morphology following rod death in 5-7 dpf larvae . No studies were performed on adult
[137]
animals. The use of the MTZ/NTR system enables investigators to selectively ablate rods in an inducible
manner. The ability to trigger acute damage to a specific cell type at a pre-determined time allows
investigators to compare the regenerative response between acute injury and chronic degeneration models.

Tg(rho:Mmu.Rho_P23H-FLAG)uth4Tg
Transgenic models of adRP that express mutant forms of rhodopsin in rod photoreceptors have been made