An efficient inducible RPE-Selective cre transgenic mouse line

Chen, Ming; Kim, Lily; Lu, Carolyn; Zeng, Hong; Vollrath, Douglas

doi:10.1016/j.exer.2020.108370

Cited by 8 publications

(11 citation statements)

References 38 publications

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“…This is consistent with preservation of reduced ERG expression in a subset of KCNJ13CKO ko/ko, VMD2-Cre +/+ mice. In addition, it has been shown recently that Cre expression with this model shows some leakiness with expression in other retinal cells than the RPE ( Chen et al, 2021 ; Choi et al, 2021 ). This is also apparent in Figure 5B , in which Cre expression is seen not only in the RPE but also the GCL and INL.…”

Section: Discussionmentioning

confidence: 83%

Retinal Development and Pathophysiology in Kcnj13 Knockout Mice

Jiao

Lei

et al. 2022

Front. Cell Dev. Biol.

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Purpose: We constructed and characterized knockout and conditional knockout mice for KCNJ13, encoding the inwardly rectifying K+ channel of the Kir superfamily Kir7.1, mutations in which cause both Snowflake Vitreoretinal Degeneration (SVD) and Retinitis pigmentosa (RP) to further elucidate the pathology of this disease and to develop a potential model system for gene therapy trials.Methods: A Kcnj13 knockout mouse line was constructed by inserting a gene trap cassette expressing beta-galactosidase flanked by FRT sites in intron 1 with LoxP sites flanking exon two and converted to a conditional knockout by FLP recombination followed by crossing with C57BL/6J mice having Cre driven by the VMD2 promoter. Lentiviral replacement of Kcnj13 was driven by the EF1a or VMD2 promoters.Results: Blue-Gal expression is evident in E12.5 brain ventricular choroid plexus, lens, neural retina layer, and anterior RPE. In the adult eye expression is seen in the ciliary body, RPE and choroid. Adult conditional Kcnj13 ko mice show loss of photoreceptors in the outer nuclear layer, inner nuclear layer thinning with loss of bipolar cells, and thinning and disruption of the outer plexiform layer, correlating with Cre expression in the overlying RPE which, although preserved, shows morphological disruption. Fundoscopy and OCT show signs of retinal degeneration consistent with the histology, and photopic and scotopic ERGs are decreased in amplitude or extinguished. Lentiviral based replacement of Kcnj13 resulted in increased ERG c- but not a- or b- wave amplitudes.Conclusion: Ocular KCNJ13 expression starts in the choroid, lens, ciliary body, and anterior retina, while later expression centers on the RPE with no/lower expression in the neuroretina. Although KCNJ13 expression is not required for survival of the RPE, it is necessary for RPE maintenance of the photoreceptors, and loss of the photoreceptor, outer plexiform, and outer nuclear layers occur in adult KCNJ13 cKO mice, concomitant with decreased amplitude and eventual extinguishing of the ERG and signs of retinitis pigmentosa on fundoscopy and OCT. Kcnj13 replacement resulting in recovery of the ERG c- but not a- and b-waves is consistent with the degree of photoreceptor degeneration seen on histology.

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Section: Discussionmentioning

confidence: 83%

Retinal Development and Pathophysiology in Kcnj13 Knockout Mice

Jiao

Lei

et al. 2022

Front. Cell Dev. Biol.

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“…We combined a conditional (floxed) allele of the intraflagellar transport protein (Ift88), which is essential for cilia formation and maintenance, with a Cre allele-driven by the BEST1 promoter to drive RPE-specific cilia loss. The BEST1-Cre transgene drives ocular Cre expression that begins postnatally at about P10 70 , 71 . The mosaic expression of the BEST1-Cre in the RPE allowed us to simultaneously assess the impact of cilia loss on RPE and on control neighboring cells in the retina.…”

Section: Resultsmentioning

confidence: 99%

Cilia-associated wound repair mediated by IFT88 in retinal pigment epithelium

Ning

Bhuckory

et al. 2023

Sci Rep

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Primary cilia are conserved organelles that integrate extracellular cues into intracellular signals and are critical for diverse processes, including cellular development and repair responses. Deficits in ciliary function cause multisystemic human diseases known as ciliopathies. In the eye, atrophy of the retinal pigment epithelium (RPE) is a common feature of many ciliopathies. However, the roles of RPE cilia in vivo remain poorly understood. In this study, we first found that mouse RPE cells only transiently form primary cilia. We then examined the RPE in the mouse model of Bardet-Biedl Syndrome 4 (BBS4), a ciliopathy associated with retinal degeneration in humans, and found that ciliation in BBS4 mutant RPE cells is disrupted early during development. Next, using a laser-induced injury model in vivo, we found that primary cilia in RPE reassemble in response to laser injury during RPE wound healing and then rapidly disassemble after the repair is completed. Finally, we demonstrated that RPE-specific depletion of primary cilia in a conditional mouse model of cilia loss promoted wound healing and enhanced cell proliferation. In summary, our data suggest that RPE cilia contribute to both retinal development and repair and provide insights into potential therapeutic targets for more common RPE degenerative diseases.

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“…With one exception, these reported Cre lines were generated using random-integration transgenic approaches, as opposed to the targeted KI strategy used in this work, and are therefore susceptible to transgene epigenetic silencing and consequent mosaic Cre expression ( 38 ). Recently, a BEST1 -CreER T2 mouse model was reported in which a single copy of the transgene was targeted to the Rosa26 locus in an attempt to avoid this silencing issue ( 31 ). Despite an improvement in recombination efficiency, Cre activity remained somewhat mosaic in these mice, suggesting that this property could be intrinsic to the BEST1 promoter.…”

Section: Discussionmentioning

confidence: 99%

“…Although a recently described BEST1-CreER T2 mouse line (31) showed improved recombination efficiency and RPE selectivity as compared to prior inducible lines, it still did not achieve complete and specific recombination within the RPE. Hence, a critical need remains for a reliable, non-toxic, RPE-specific Cre mouse line that can achieve quantitative recombination of floxed alleles in a temporally controlled manner.…”

Section: Introductionmentioning

confidence: 87%

“…All of these Cre mouse lines suffer from various inadequacies that limit their usefulness, including mosaic patterns of Cre expression in the RPE, inducer-independent (i.e., “leaky”) Cre activity, off-target Cre expression, and, in the case of some constitutively active systems, Cre-mediated toxicity to the RPE ( 29 , 30 ). Although a recently described BEST1 -CreER T2 mouse line ( 31 ) showed improved recombination efficiency and RPE selectivity as compared with prior inducible lines, it still did not achieve complete and specific recombination within the RPE. Hence, a critical need remains for a reliable, nontoxic, RPE-specific Cre mouse line that can achieve quantitative recombination of floxed alleles in a temporally controlled manner.…”

Section: Introductionmentioning

confidence: 94%

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An inducible Cre mouse for studying roles of the RPE in retinal physiology and disease

Choi¹,

Suh²,

Einstein³

et al. 2021

JCI Insight

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The retinal pigment epithelium (RPE) provides vital metabolic support for retinal photoreceptor cells and also is an important player in numerous retinal diseases. Gene manipulation in mice using the Cre-LoxP system is an invaluable tool for studying the genetic basis of these retinal diseases. However, existing RPE-targeted Cre mouse lines have critical limitations that restrict their reliability for studies of disease pathogenesis and treatment, including mosaic Cre expression, inducer-independent activity, off-target Cre expression, and intrinsic toxicity. Here, we report the generation and characterization of a knock-in mouse line in which a P2A-CreER T2 coding sequence is fused with the native RPE-specific 65 kDa protein (Rpe65) gene for co-translational expression of CreER T2 . Cre +/mice were able to recombine a stringent Cre reporter allele with >99% efficiency and absolute RPE specificity upon tamoxifen induction at both post-natal days (PD) 21 and 50. Tamoxifen-independent Cre activity was negligible at PD64. Moreover, tamoxifen-treated Cre +/mice displayed no signs of structural or functional retinal pathology up to 4 months of age. Despite weak RPE65 expression from the knock-in allele, visual cycle function was normal in Cre +/mice. These data indicate that Rpe65 CreERT2 mice are wellsuited for studies of gene function and pathophysiology in the RPE.

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An efficient inducible RPE-Selective cre transgenic mouse line

Cited by 8 publications

References 38 publications

Retinal Development and Pathophysiology in Kcnj13 Knockout Mice

Retinal Development and Pathophysiology in Kcnj13 Knockout Mice

Cilia-associated wound repair mediated by IFT88 in retinal pigment epithelium

An inducible Cre mouse for studying roles of the RPE in retinal physiology and disease

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