One-step Generation of Mice Carrying a Conditional Allele Together with an HA-tag Insertion for the Delta Opioid Receptor

Overview

Journal Sci Rep

Specialty Science

Date 2017 Mar 17

PMID 28300205

Citations 8

Authors

Dongru Su

Min Wang

Chenli Ye

Jiahui Fang

Yanhui Duan

Zhenghong Zhang

Qiuhong Hua

Changjie Shi

Lihong Zhang

Ru Zhang

Xin Xie

Affiliations

Soon will be listed here.

Abstract

G protein-coupled receptors (GPCRs) are important modulators of many physiological functions and excellent drug targets for many diseases. However, to study the functions of endogenous GPCRs is still a challenging task, partially due to the low expression level of GPCRs and the lack of highly potent and selective GPCR antibodies. Overexpression or knock-in of tagged GPCRs, or knockout of specific GPCRs in mice, are common strategies used to study the in vivo functions of these receptors. However, generating separate mice carrying tagged GPCRs or conditional alleles for GPCRs is labor intensive, and requires additional breeding costs. Here we report the generation of mice carrying an HA-tagged DOR (delta opioid receptor) flanked by LoxP sequences at the endogenous DOR locus using a single recombination step, aided by the TALEN system. These animals can be used directly to study the expression, localization, protein-protein interaction and signal transduction of endogenous DOR using anti-HA antibodies. By crossing with mice expressing tissue-specific Cre, these mice can also generate offspring with DOR knockout within specific tissues. These mice are powerful tools to study the in vivo functions of DOR. Furthermore, the gene modification strategy could also be used to study the functions of many other GPCRs.

Citing Articles

Knock-In Mouse Models to Investigate the Functions of Opioid Receptors .

Degrandmaison J, Rochon-Hache S, Parent J, Gendron L Front Cell Neurosci. 2022; 16:807549.

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Identification and Expression Analysis of G Protein-Coupled Receptors in the Miridae Insect .

Gao H, Li Y, Wang M, Song X, Tang J, Feng F Front Endocrinol (Lausanne). 2021; 12:773669.

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HA-MOP knockin mice express the canonical µ-opioid receptor but lack detectable splice variants.

Fritzwanker S, Mouledous L, Mollereau C, Froment C, Burlet-Schiltz O, Effah F Commun Biol. 2021; 4(1):1070.

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In vivo mapping of a GPCR interactome using knockin mice.

Degrandmaison J, Abdallah K, Blais V, Genier S, Lalumiere M, Bergeron F Proc Natl Acad Sci U S A. 2020; 117(23):13105-13116.

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References

Ballesteros J, Palczewski K . G protein-coupled receptor drug discovery: implications from the crystal structure of rhodopsin. Curr Opin Drug Discov Devel. 2003; 4(5):561-74. PMC: 1383658. View

Orr A, Hsiao E, Wang M, Ho K, Kim D, Wang X . Astrocytic adenosine receptor A2A and Gs-coupled signaling regulate memory. Nat Neurosci. 2015; 18(3):423-34. PMC: 4340760. DOI: 10.1038/nn.3930. View

Wess J, Eglen R, Gautam D . Muscarinic acetylcholine receptors: mutant mice provide new insights for drug development. Nat Rev Drug Discov. 2007; 6(9):721-33. DOI: 10.1038/nrd2379. View

Michel M, Wieland T, Tsujimoto G . How reliable are G-protein-coupled receptor antibodies?. Naunyn Schmiedebergs Arch Pharmacol. 2009; 379(4):385-8. DOI: 10.1007/s00210-009-0395-y. View

Gautam D, Han S, Hamdan F, Jeon J, Li B, Li J . A critical role for beta cell M3 muscarinic acetylcholine receptors in regulating insulin release and blood glucose homeostasis in vivo. Cell Metab. 2006; 3(6):449-61. DOI: 10.1016/j.cmet.2006.04.009. View

Shalem O, Sanjana N, Zhang F . High-throughput functional genomics using CRISPR-Cas9. Nat Rev Genet. 2015; 16(5):299-311. PMC: 4503232. DOI: 10.1038/nrg3899. View

Shenoy S, Lefkowitz R . Trafficking patterns of beta-arrestin and G protein-coupled receptors determined by the kinetics of beta-arrestin deubiquitination. J Biol Chem. 2003; 278(16):14498-506. DOI: 10.1074/jbc.M209626200. View

Tanaka S, Ishii K, Kasai K, Yoon S, Saeki Y . Neural expression of G protein-coupled receptors GPR3, GPR6, and GPR12 up-regulates cyclic AMP levels and promotes neurite outgrowth. J Biol Chem. 2007; 282(14):10506-15. DOI: 10.1074/jbc.M700911200. View

Ozawa A, Brunori G, Mercatelli D, Wu J, Cippitelli A, Zou B . Knock-In Mice with NOP-eGFP Receptors Identify Receptor Cellular and Regional Localization. J Neurosci. 2015; 35(33):11682-93. PMC: 4540802. DOI: 10.1523/JNEUROSCI.5122-14.2015. View

10.

Persson A, Thorlin T, Bull C, Zarnegar P, Ekman R, Terenius L . Mu- and delta-opioid receptor antagonists decrease proliferation and increase neurogenesis in cultures of rat adult hippocampal progenitors. Eur J Neurosci. 2003; 17(6):1159-72. DOI: 10.1046/j.1460-9568.2003.02538.x. View

11.

Rosenbaum D, Rasmussen S, Kobilka B . The structure and function of G-protein-coupled receptors. Nature. 2009; 459(7245):356-63. PMC: 3967846. DOI: 10.1038/nature08144. View

12.

Gaj T, Gersbach C, Barbas 3rd C . ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol. 2013; 31(7):397-405. PMC: 3694601. DOI: 10.1016/j.tibtech.2013.04.004. View

13.

Lewandoski M . Conditional control of gene expression in the mouse. Nat Rev Genet. 2001; 2(10):743-55. DOI: 10.1038/35093537. View

14.

Barker N, van Es J, Kuipers J, Kujala P, van den Born M, Cozijnsen M . Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature. 2007; 449(7165):1003-7. DOI: 10.1038/nature06196. View

15.

Luttrell L, Ferguson S, Daaka Y, Miller W, Maudsley S, Della Rocca G . Beta-arrestin-dependent formation of beta2 adrenergic receptor-Src protein kinase complexes. Science. 1999; 283(5402):655-61. DOI: 10.1126/science.283.5402.655. View

16.

Luker K, Gupta M, Luker G . Imaging CXCR4 signaling with firefly luciferase complementation. Anal Chem. 2008; 80(14):5565-73. PMC: 5258185. DOI: 10.1021/ac8005457. View

17.

Pradhan A, Smith M, McGuire B, Evans C, Walwyn W . Chronic inflammatory injury results in increased coupling of delta opioid receptors to voltage-gated Ca2+ channels. Mol Pain. 2013; 9:8. PMC: 3621800. DOI: 10.1186/1744-8069-9-8. View

18.

Shenoy S, McDonald P, Kohout T, Lefkowitz R . Regulation of receptor fate by ubiquitination of activated beta 2-adrenergic receptor and beta-arrestin. Science. 2001; 294(5545):1307-13. DOI: 10.1126/science.1063866. View

19.

Liu S, Umezu-Goto M, Murph M, Lu Y, Liu W, Zhang F . Expression of autotaxin and lysophosphatidic acid receptors increases mammary tumorigenesis, invasion, and metastases. Cancer Cell. 2009; 15(6):539-50. PMC: 4157573. DOI: 10.1016/j.ccr.2009.03.027. View

20.

Kos C . Cre/loxP system for generating tissue-specific knockout mouse models. Nutr Rev. 2004; 62(6 Pt 1):243-6. DOI: 10.1301/nr2004.jun243-246. View