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Magdalena Maria Staniszewska, Daniel Taub, Eric Pierce, Qin Liu; Deficiency of tetratricopeptide repeat domain 21B (Ttc21B) leads to early onset of photoreceptor cell death. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4388.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in TTC21B gene, which encodes the intraflagellar transport protein 139 (IFT139), cause ciliopathy that involves retinal degeneration and other disorders related to cilia dysfunction. The goal was to use Ttc21b conditional knockout mice to investigate the function of Ttc21b and retrograde IFT in photoreceptor sensory cilia (PSC), and the mechanism by which mutations in TTC21B cause photoreceptor cell death.
The embryos of mice with a targeted allele, Ttc21btm1a(KOMP)Wtsi were obtained from the KOMP project. These mice were crossed with Flpe deleter mice to remove the reporter/selection cassette, and generate a conditional Ttc21bflox allele with exon 2 of Ttc21b floxed. The Ttc21bflox/flox mice were crossed with Rhodopsin-iCre transgenic mice (iCre-75) to generate mice that lack Ttc21b in rod photoreceptor cells. Retinas from Ttc21bflox/flox/Rho-iCre+ conditional mutant mice (CKO) and Ttc21bwt/wt/Rho-iCre+ (WT) were collected for analyses. The progress of photoreceptor degeneration was assessed by immunostaining, quantification of the outer nuclear layer (ONL) thickness, and TUNEL assay.
Knockout of Ttc21b expression was achieved in Cre-positive rods, evidenced by a lack of Ttc21b labeling in the retina from CKO mice. Photoreceptor cells in CKO mice retina underwent early onset cell death, with a significant number of TUNEL-positive cells detected as early as age P11. The degeneration progressed rapidly with only 2-3 rows of photoreceptor cells left at 1 month, down to 1 row of cones in 3 months old CKO mice. Co-staining of the Rp1 showed that the deficiency of Ttc21b does not affect the axonemal structure of PSC. Co-staining with rhodopsin indicated that abnormal outer segments were formed at P11. At 1 month the majority of rhodopsin was mis-localized to the inner segments of photoreceptor cells, with no outer segments observed in the CKO mice retinas.
Conditional knockout of Ttc21b in rods results in early onset of cell death, associated with defective PSC formation. This implies that TTC21B, as a retrograde IFT protein, is required for normal PSC formation and photoreceptor cell survival. Future work will investigate the role of retrograde IFT in photoreceptor biology, and the mechanisms by which mutations in TTC21B gene cause ciliopathy.
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