Dataset Information

One-step generation of mice carrying a conditional allele together with an HA-tag insertion for the delta opioid receptor.

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.

SUBMITTER: Su D

PROVIDER: S-EPMC5353682 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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One-step generation of mice carrying a conditional allele together with an HA-tag insertion for the delta opioid receptor.

Su Dongru D Wang Min M Ye Chenli C Fang Jiahui J Duan Yanhui Y Zhang Zhenghong Z Hua Qiuhong Q Shi Changjie C Zhang Lihong L Zhang Ru R Xie Xin X

Scientific reports 20170316

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 ...[more]

PMID: 28300205

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Project description:Extracellular vesicles (EVs) are a unique mode of intercellular communication capable of specificity in transmitting signals and cargo to coordinate local and distant cellular functions. A key example of this is the essential role that EVs secreted by epithelial cells lining the lumen of the male reproductive tract play in post-spermatogenic sperm maturation. We recently showed in a preclinical mouse model that this fundamental process had a causal role in somatic-to-germline transmission of biological information regarding prior stress experience capable of altering the rate of fetal development. However, critical mechanistic questions remain unanswered as to the processes by which signaling occurs between EVs and sperm, and whether EVs or their cargo are delivered at conception and are detectable in the early embryo. Unfortunately, notable methodological limitations shared across EV biology, particularly in the isolation and labeling of EVs, complicate efforts to answer these important questions as well as questions on EV targeting specificity and mechanisms. In our current studies, we developed a novel approach to track EVs using a conditional transgenic construct designed to label EVs via conditional Cre-induced hemagglutinin (HA) tagging of the EV endogenous tetraspanin, CD63. In our exhaustive validation steps, this internal small molecular weight tag did not affect EV secretion or functionality, a common problem found in the previous design of EV tags using larger molecular weight proteins, including fluorescent proteins. Utilizing a stably transfected immortalized epididymal epithelial cell line, we first validated key parameters of the conditional HA-tagged protein packaged into secreted EVs. Importantly, we systematically confirmed that expression of the CD63-HA had no impact on the production, size distribution, or surface charge of secreted EVs, nor did it alter the tetraspanin or miRNA composition of these EVs. We also utilized the CD63-HA EVs to verify physical interactions with sperm. Finally, using in vitro fertilization we produced some of the first images confirming sperm delivered EV cargo at conception and still detectable in the early-stage embryo. As such, this construct serves as a methodological advance and as a valuable tool, with applications in the study of EV function across biomedical research areas.

Dataset Information

One-step generation of mice carrying a conditional allele together with an HA-tag insertion for the delta opioid receptor.

Publications

One-step generation of mice carrying a conditional allele together with an HA-tag insertion for the delta opioid receptor.

Similar Datasets

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