June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Inducible gene targeting in mouse retinal neurons expressing Grm6
Author Affiliations & Notes
  • Yu-Jiun Chen
    Ophthalmology, Baylor College of Medicine, Houston, Texas, United States
  • Hoon Shim
    Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, Virginia, United States
  • Crystal S. Shin
    Ophthalmology, Baylor College of Medicine, Houston, Texas, United States
  • Ghanashyam Acharya
    Ophthalmology, Baylor College of Medicine, Houston, Texas, United States
  • Ching-Kang Jason Chen
    Ophthalmology, Baylor College of Medicine, Houston, Texas, United States
    Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas, United States
  • Footnotes
    Commercial Relationships   Yu-Jiun Chen, None; Hoon Shim, None; Crystal Shin, None; Ghanashyam Acharya, None; Ching-Kang Chen, None
  • Footnotes
    Support  NIH Grants EY013811, EY022228, EY002520, unrestrcted grant from Research to Prevent Blindness, Retina Research Foundation
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 2220. doi:
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    • Get Citation

      Yu-Jiun Chen, Hoon Shim, Crystal S. Shin, Ghanashyam Acharya, Ching-Kang Jason Chen; Inducible gene targeting in mouse retinal neurons expressing Grm6. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2220.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : The purpose of the study is to generate mouse lines for inducible gene targeting in depolarizing bipolar cells (DBCs).

Methods : Three engineered transgenic constructs with iCre, mCherry, and ERT2-Cre-ERT2 (ECE) cDNAs downstream of a 10 kb mouse Grm6 promoter fragment were made, respectively. The iCre and mCherry constructs were mixed and injected pronuclearly into embryos of C57BL/6 and Balb/c mixed background and one founder line, MCV11F, was established. The ECE construct was injected alone and one founder line, T1, was established. Cre activity was revealed by commercial Z/EP or Ai9 reporter lines. Cre induction in the T1/Ai9 line was done by intraperitoneal injections of varying doses of tamoxifen (TAM) or by topical application of 4-hydroxytamoxifen (4-HT) loaded nanowafers on the cornea.

Results : The MCV11F line has robust mCherry and Cre recombinase expression in all adult DBCs. However, in the Z/EP background, we found GFP expression throughout the entire retina, indicating that Cre was expressed earlier during development. Without induction, the T1/Ai9 line displays Cre activity in ~50 neurons in young adults and ~100 in mice over one year of age. Intraperitoneal delivery of 500 nmol per gram body weight per day of TAM over five days is sufficient to induce Cre activity in >90% of DBCs. By titrating the amount of TAM injected, a single delivery of 10-25 nmol per gram body weight could induce tdTomato expression in ~300 intermittently dispersed neurons per retina. To induce Cre activity in the T1 mice unilaterally, we employed a nanowafer delivery system of 4-HT at the dose of (5 µg/wafer/day). This could also induce reporter expression between 200-1000 DBCs in the treated eye. Under inductive conditions where 100 to 1000 DBCs expressed tdTomato, examination of the axonal stratification levels of ~200 cells in the inner plexiform layer (IPL) showed that rod bipolar cells (RBCs) and all known cone ON bipolar cells except the type-9 (CB9) could be labeled. Interestingly, we encounter a few cells whose axons stratify to the OFF center, suggesting that certain hyperpolarizing bipolar cells may also express GRM6.

Conclusions : Despite being a good marker line, the precarious Cre expression during development of MCV11F mice restricts its use. The T1 line is more suitable for inducible gene targeting in DBCs. Its utility may be expanded by further exploration of the type, amount, and routes of reagents used for induction.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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