Ophthalmic Administration of a DNA Plasmid Harboring the Murine Tph2 Gene: Evidence of Recombinant Tph2-FLAG in Brain Structures
| Autor: | Francisco Xavier Guerra-Castillo, Leticia Manuel-Apolinar, Charmina Aguirre-Alvarado, Sandra Orozco-Suárez, Miguel Pérez de la Mora, Vilma Carolina Bekker-Méndez, Norma Oviedo, Gloria Martínez-Pérez, Emiliano Tesoro-Cruz |
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| Rok vydání: | 2020 |
| Předmět: |
0106 biological sciences
Male Recombinant Fusion Proteins Gene Expression Bioengineering Administration Ophthalmic Biology Tryptophan Hydroxylase 01 natural sciences Applied Microbiology and Biotechnology Biochemistry law.invention 03 medical and health sciences Mice Plasmid In vivo law 010608 biotechnology Animals Humans Molecular Biology Gene 030304 developmental biology 0303 health sciences Mice Inbred BALB C TPH2 Raphe Optic Nerve Molecular biology In vitro HEK293 Cells Blood-Brain Barrier Recombinant DNA Serotonin Biotechnology Plasmids |
| Zdroj: | Molecular biotechnology. 62(3) |
| ISSN: | 1559-0305 |
| Popis: | Tryptophan hydroxylase-type 2 (Tph2) is the first rate-limiting step in the biosynthesis of serotonin (5-HT) in the brain. The ophthalmic administration (Op-Ad) is a non-invasive method that allows delivering genetic vehicles through the eye and reaches the brain. Here, the murine Tph2 gene was cloned in a non-viral vector (pIRES-hrGFP-1a), generating pIRES-hrGFP-1a-Tph2, plus the FLAG-tag. Recombinant Tph2-FLAG was detected and tested in vitro and in vivo, where 25 μg of pIRES-hrGFP-1a-Tph2-FLAG was Op-Ad to mice. The construct was capable of expressing and producing the recombinant Tph2-FLAG in vitro and in vivo. The in vivo assays showed that the construct efficiently crossed the Hemato-Ocular Barrier and the Blood–Brain Barrier, reached brain cells, passed the optical nerves, and transcribed mRNA-Tph2-FLAG in different brain areas. The recombinant Tph2-FLAG was observed in amygdala and brainstem, mainly in raphe dorsal and medial. Relative Tph2 expression of threefold over basal level was recorded three days after Op-Ad. These results demonstrated that pIRES-hrGFP-Tph2-FLAG, administrated through the eyes was capable of reaching the brain, transcribing, and translating Tph2. In conclusion, this study showed the feasibility of delivering therapeutic genes, such as the Tph2, the first enzyme, rate-limiting step in the 5-HT biosynthesis. |
| Databáze: | OpenAIRE |
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