Highly Selective Transgene Expression Through Double-Floxed Inverted Orientation System by Using a Unilateral Spacer Sequence

Matsushita, Natsuki; Kato, Shigekí; Nishizawa, Kayo; Sugawara, Masateru; Takeuchi, Kosei; Miyasaka, Yoshiki; Mashimo, Tomoji; Kobayashi, Kazuto

doi:10.2139/ssrn.4083378

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“…In the present study, as the flip/excision switch (FLEX) system we used the transfer plasmid containing the gene cassette encoding green fluorescent protein (GFP; GenBank accession number L29345; Inouye and Tsuji, 1994) in the inverted orientation, which is flanked by a pair of double recognition sites consisting of lox P and lox 2272 sites in the opposite direction with an alternate order at both ends of the cassette. Another gene cassette encoding TurboFP635 (GenBank accession number AB982101; Takai et al, 2015) was inserted between two recognition sites at the 3’-end of the GFP sequence to protect non-specific expression of transgene in the tissues after the injection of AAV-FLEX-GFP vector (Matsushita et al, 2022 [bioRxiv]). The crude viral lysate was purified with two rounds of CsCl gradient ultracentrifugation at 100,000× g and 4°C for 23 h using 13.5-mL Quick-Seal tubes and a NVT65 rotor (Beckman Coulter, Brea, CA).…”

Section: Methodsmentioning

confidence: 99%

Catecholaminergic cell type-specific expression of Cre recombinase in knock-in transgenic rats generated by the Combi-CRISPR technology

Matsushita

Nishizawa

Kato

et al. 2022

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Background: Cell groups containing catecholamines provide a useful model to study the molecular and cellular mechanisms underlying the morphogenesis, physiology, and pathology of the central nervous system. For this purpose, it is necessary to establish a system to induce catecholaminergic group-specific expression of Cre recombinase. Recently, we introduced a gene cassette encoding 2A peptide fused to Cre recombinase into the site between the C-terminus and translational termination codons of the rat tyrosine hydroxylase (TH) open reading frame by the Combi-CRISPR technology, which is a genomic editing method to enable an efficient knock-in (KI) of long DNA sequence into a target site. However, the expression patterns of the transgene and its function as well as the effect of the mutation on the biochemical and behavioral phenotypes in the KI strains have not been characterized yet. New Method: We aimed to evaluate the usefulness of TH-Cre KI rats as an experimental model for investigating the structure and function of catecholaminergic neurons in the brain. Results: We detected cell type-specific expression of Cre recombinase and site-specific recombination activity in the representative catecholaminergic groups in the TH-Cre KI rat strains. In addition, we measured TH expression level and catecholamine accumulation in the brain regions, and spontaneous locomotion, indicating that catecholamine metabolism and general behavior are apparently normal in these KI rats. Conclusions: TH-Cre KI rat strains produced by the Combi-CRISPR system offer a beneficial model to study the molecular and cellular mechanics for the morphogenesis, physiology, and pathology of catecholamine-containing neurons in the brain.

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Section: Methodsmentioning

confidence: 99%