Spatiotemporal control of genome engineering in cone photoreceptors

Nan-Kai Wang; Pei-Kang Liu; Yang Kong; Yun-Ju Tseng; Laura A Jenny; Nicholas D Nolan; Nelson Chen; Hung-Hsi Wang; Chun Wei Hsu; Wan-Chun Huang; Janet R Sparrow; Chyuan-Sheng Lin; Stephen H Tsang

doi:10.1186/s13578-023-01033-3

Spatiotemporal control of genome engineering in cone photoreceptors

Cell Biosci. 2023 Jun 28;13(1):119. doi: 10.1186/s13578-023-01033-3.

Authors

Nan-Kai Wang^#^{1

2}, Pei-Kang Liu^#^{3

4

5

6}, Yang Kong³, Yun-Ju Tseng³, Laura A Jenny³, Nicholas D Nolan^{3

7}, Nelson Chen^{3

8}, Hung-Hsi Wang^{3

9}, Chun Wei Hsu³, Wan-Chun Huang³, Janet R Sparrow¹⁰, Chyuan-Sheng Lin^{11

12}, Stephen H Tsang^{13

14}

Affiliations

¹ Edward S. Harkness Eye Institute, Department of Ophthalmology, Columbia University Irving Medical Center, New York, NY, 10032, USA. wang.nankai@gmail.com.
² Vagelos College of Physicians and Surgeons, Columbia University, New York, USA. wang.nankai@gmail.com.
³ Edward S. Harkness Eye Institute, Department of Ophthalmology, Columbia University Irving Medical Center, New York, NY, 10032, USA.
⁴ Department of Ophthalmology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan.
⁵ School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
⁶ Institute of Biomedical Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan.
⁷ Department of Biomedical Engineering, The Fu Foundation School of Engineering and Applied Science, Columbia University, New York, NY, 10027, USA.
⁸ Faculty of Health Sciences, Queen's University, Kingston, ON, Canada.
⁹ College of Arts and Sciences, University of Miami, Coral Gables, FL, USA.
¹⁰ Departments of Ophthalmology, Pathology and Cell Biology, Columbia University, New York, USA.
¹¹ Department of Pathology & Cell Biology, Columbia University Irving Medical Center, New York, NY, 10032, USA.
¹² Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, NY, 10032, USA.
¹³ Edward S. Harkness Eye Institute, Department of Ophthalmology, Columbia University Irving Medical Center, New York, NY, 10032, USA. sht2@cumc.columbia.edu.
¹⁴ Jonas Children's Vision Care, and Bernard and Shirlee Brown Glaucoma Laboratory, Columbia Stem Cell Initiative, Departments of Ophthalmology, Pathology and Cell Biology, Institute of Human Nutrition, Vagelos College of Physicians and Surgeons, Columbia University, New York, USA. sht2@cumc.columbia.edu.

^# Contributed equally.

Abstract

Background: Cones are essential for color recognition, high resolution, and central vision; therefore cone death causes blindness. Understanding the pathophysiology of each cell type in the retina is key to developing therapies for retinal diseases. However, studying the biology of cone cells in the rod-dominant mammalian retina is particularly challenging. In this study, we used a bacterial artificial chromosome (BAC) recombineering method to knock in the "CreER^T2" sequence into the Gnat2 and Arr3 genes, respectively and generated three novel inducible CreER^T2 mice with different cone cell specificities.

Results: These models (Gnat2^CreERT2, Arr3^T2ACreERT2, and Arr3^P2ACreERT2) express temporally controllable Cre recombinase that achieves conditional alleles in cone photoreceptors. Cre-LoxP recombination can be induced as early as postnatal day (PD) two upon tamoxifen injection at varying efficiencies, ranging from 10 to 15% in Gnat2^CreERT2, 40% in Arr3^T2ACreERT2, and 100% in Arr3^P2ACreERT2. Notably, knocking in the P2A-CreERT2 cassette does not affect cone cell morphology and functionality. Most cone-phototransduction enzymes, including Opsins, CNGA3, etc. are not altered except for a reduction in the Arr3 transcript.

Conclusions: The Arr3^P2ACreERT2 mouse, an inducible cone-specific Cre driver, is a valuable line in studying cone cell biology, function, as well as its relationship with rod and other retinal cells. Moreover, the Cre activity can be induced by delivering tamoxifen intragastrically as early as PD2, which will be useful for studying retinal development or in rapid degenerative mouse models.

Keywords: 2A peptide; Arrestin 3; Cone arrestin; Cre-LoxP recombination; CreERT2.

Abstract

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