GRK5 knockout mice created by TALEN‐mediated gene targeting (576.2)

Abstract

G protein‐coupled receptor kinase 5 (GRK5) is one of the seven GRK family members whose primary function is to desensitize G protein‐coupled receptors (GPCRs). In recently, GRK5 deficiency has been linked to early Alzheimer disease (AD), but the mechanism by which GRK5 deficiency may accelerate to AD pathogenesis remains elusive. The GRK5 mRNA is expressed widely in brain and peripheral tissues, with highest expression evident heart, lung, and placenta. In cellular model systems, GRK5 can phosphorylate several neuronal GPCRs including ß2 adrenergic, M2‐muscarinic, secretin, angiotensin AT1, and thyroid stimulating hormone receptors. Transcription activator‐like effector nucleases (TALENs) are programmable nucleases that join FokI endonucleases with the modular DNA‐binding domain of TALEs and highly effective in inducing mutations at specific genome loci. TALEN‐mediated mutagenesis in zygotes is a potential alternative to conventional gene targeting in mice. In the presented study, we report the generation of mice with genetic knockout of the GRK gene using TALENs. We designed TALEN vectors for exon 1, 3 and 5 of mouse GRK5 gene and tested their ability to alter the each surrogate vector in 293T cells. We prepared of mRNAs for the linearized TALEN using the mMessage mMachine T7 Ultra kit. mRNAs (4ng/μl) was injected into cytoplasm of 180 one‐cell embryos. After incubation for 24 hours, the selected two‐cell embryos transferred into the oviduct of seven pseudopregnant C57BL/6 mice. We confirmed the genotype of Fo mice by sequencing and T7E1 assay.Grant Funding Source: Supported by BK 21 plus program, the Ministry of Education, Science and Technology, Korea