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Ophthalmic Administration of a DNA Plasmid Harboring the Murine Tph2 Gene: Evidence of Recombinant Tph2-FLAG in Brain Structures
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 clon...
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| Published in: | Molecular biotechnology 2020-03, Vol.62 (3), p.200-209 |
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| creator | Tesoro-Cruz, Emiliano Oviedo, Norma Manuel-Apolinar, Leticia Orozco-Suárez, Sandra Pérez de la Mora, Miguel Martínez-Pérez, Gloria Guerra-Castillo, Francisco Xavier Aguirre-Alvarado, Charmina Bekker-Méndez, Vilma Carolina |
| description | 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. |
| doi_str_mv | 10.1007/s12033-020-00239-x |
| format | article |
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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.</description><identifier>ISSN: 1073-6085</identifier><identifier>EISSN: 1559-0305</identifier><identifier>DOI: 10.1007/s12033-020-00239-x</identifier><identifier>PMID: 32030628</identifier><language>eng</language><publisher>New York: Springer US</publisher><subject>Administration, Ophthalmic ; Amygdala ; Animals ; Biochemistry ; Biological Techniques ; Biosynthesis ; Biotechnology ; Blood-brain barrier ; Blood-Brain Barrier - cytology ; Blood-Brain Barrier - metabolism ; Brain ; Brain stem ; Cell Biology ; Chemistry ; Chemistry and Materials Science ; Constraining ; Deoxyribonucleic acid ; DNA ; Eye (anatomy) ; Feasibility studies ; Flags ; Gene Expression ; HEK293 Cells ; Human Genetics ; Humans ; Hydroxylase ; Male ; Mice ; Mice, Inbred BALB C ; mRNA ; Nerves ; Optic Nerve - cytology ; Optic Nerve - metabolism ; Original Paper ; Plasmids - genetics ; Plasmids - pharmacokinetics ; Plasmids - pharmacology ; Protein Science ; Recombinant ; Recombinant Fusion Proteins - biosynthesis ; Recombinant Fusion Proteins - genetics ; Serotonin ; Tryptophan ; Tryptophan hydroxylase ; Tryptophan Hydroxylase - biosynthesis ; Tryptophan Hydroxylase - genetics</subject><ispartof>Molecular biotechnology, 2020-03, Vol.62 (3), p.200-209</ispartof><rights>Springer Science+Business Media, LLC, part of Springer Nature 2020</rights><rights>Molecular Biotechnology is a copyright of Springer, (2020). All Rights Reserved.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c374t-8b510fe452d6ec98fc17c0876b67fda7a50a0ba29ee708ae90ac8cc0308392733</citedby><cites>FETCH-LOGICAL-c374t-8b510fe452d6ec98fc17c0876b67fda7a50a0ba29ee708ae90ac8cc0308392733</cites><orcidid>0000-0001-8877-8439</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27924,27925</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/32030628$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Tesoro-Cruz, Emiliano</creatorcontrib><creatorcontrib>Oviedo, Norma</creatorcontrib><creatorcontrib>Manuel-Apolinar, Leticia</creatorcontrib><creatorcontrib>Orozco-Suárez, Sandra</creatorcontrib><creatorcontrib>Pérez de la Mora, Miguel</creatorcontrib><creatorcontrib>Martínez-Pérez, Gloria</creatorcontrib><creatorcontrib>Guerra-Castillo, Francisco Xavier</creatorcontrib><creatorcontrib>Aguirre-Alvarado, Charmina</creatorcontrib><creatorcontrib>Bekker-Méndez, Vilma Carolina</creatorcontrib><title>Ophthalmic Administration of a DNA Plasmid Harboring the Murine Tph2 Gene: Evidence of Recombinant Tph2-FLAG in Brain Structures</title><title>Molecular biotechnology</title><addtitle>Mol Biotechnol</addtitle><addtitle>Mol Biotechnol</addtitle><description>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.</description><subject>Administration, Ophthalmic</subject><subject>Amygdala</subject><subject>Animals</subject><subject>Biochemistry</subject><subject>Biological Techniques</subject><subject>Biosynthesis</subject><subject>Biotechnology</subject><subject>Blood-brain barrier</subject><subject>Blood-Brain Barrier - cytology</subject><subject>Blood-Brain Barrier - metabolism</subject><subject>Brain</subject><subject>Brain stem</subject><subject>Cell Biology</subject><subject>Chemistry</subject><subject>Chemistry and Materials Science</subject><subject>Constraining</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>Eye (anatomy)</subject><subject>Feasibility studies</subject><subject>Flags</subject><subject>Gene Expression</subject><subject>HEK293 Cells</subject><subject>Human Genetics</subject><subject>Humans</subject><subject>Hydroxylase</subject><subject>Male</subject><subject>Mice</subject><subject>Mice, Inbred BALB C</subject><subject>mRNA</subject><subject>Nerves</subject><subject>Optic Nerve - cytology</subject><subject>Optic Nerve - metabolism</subject><subject>Original Paper</subject><subject>Plasmids - genetics</subject><subject>Plasmids - pharmacokinetics</subject><subject>Plasmids - pharmacology</subject><subject>Protein Science</subject><subject>Recombinant</subject><subject>Recombinant Fusion Proteins - biosynthesis</subject><subject>Recombinant Fusion Proteins - genetics</subject><subject>Serotonin</subject><subject>Tryptophan</subject><subject>Tryptophan hydroxylase</subject><subject>Tryptophan Hydroxylase - biosynthesis</subject><subject>Tryptophan Hydroxylase - genetics</subject><issn>1073-6085</issn><issn>1559-0305</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2020</creationdate><recordtype>article</recordtype><recordid>eNp9kMtu2zAQRYmgQfPqD2RREN0zGZGWKHXnOrETwG2KPNYERY1qBhalkFSQ7PrppWOn3XVDzuLcO5hDyGkGZxmAPA8ZByEYcGAAXFTsZY8cZnleMRCQf0gzSMEKKPMDchTCY4KyfCI-kgORglDw8pD8vhlWcaXXnTV02nTW2RC9jrZ3tG-pphc_pvTnWofONvRK-7r31v2icYX0-5hGpPfDitMFOvxKL59tg87gJnmLpu9q67SLbwibL6cLah395nV676IfTRw9hhOy3-p1wE-7_5g8zC_vZ1dsebO4nk2XzAg5iays8wxanOS8KdBUZWsyaaCURV3IttFS56Ch1rxClFBqrECb0ph0ZikqLoU4Jl-2vYPvn0YMUT32o3dppeKikHJSSJ4gvoWM70Pw2KrB2077V5WB2jhXW-cqOVdvztVLCn3eNY91h83fyLvkBIgtEIaNPfT_Vv-n9g-2Yoxh</recordid><startdate>20200301</startdate><enddate>20200301</enddate><creator>Tesoro-Cruz, Emiliano</creator><creator>Oviedo, Norma</creator><creator>Manuel-Apolinar, Leticia</creator><creator>Orozco-Suárez, Sandra</creator><creator>Pérez de la Mora, Miguel</creator><creator>Martínez-Pérez, Gloria</creator><creator>Guerra-Castillo, Francisco Xavier</creator><creator>Aguirre-Alvarado, Charmina</creator><creator>Bekker-Méndez, Vilma Carolina</creator><general>Springer US</general><general>Springer Nature B.V</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7QL</scope><scope>7QO</scope><scope>7T7</scope><scope>7U9</scope><scope>7X7</scope><scope>7XB</scope><scope>88A</scope><scope>88E</scope><scope>88I</scope><scope>8AO</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABJCF</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>L6V</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M2P</scope><scope>M7N</scope><scope>M7P</scope><scope>M7S</scope><scope>P64</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PTHSS</scope><scope>Q9U</scope><orcidid>https://orcid.org/0000-0001-8877-8439</orcidid></search><sort><creationdate>20200301</creationdate><title>Ophthalmic Administration of a DNA Plasmid Harboring the Murine Tph2 Gene: Evidence of Recombinant Tph2-FLAG in Brain Structures</title><author>Tesoro-Cruz, Emiliano ; Oviedo, Norma ; Manuel-Apolinar, Leticia ; Orozco-Suárez, Sandra ; Pérez de la Mora, Miguel ; Martínez-Pérez, Gloria ; Guerra-Castillo, Francisco Xavier ; Aguirre-Alvarado, Charmina ; Bekker-Méndez, Vilma Carolina</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c374t-8b510fe452d6ec98fc17c0876b67fda7a50a0ba29ee708ae90ac8cc0308392733</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2020</creationdate><topic>Administration, Ophthalmic</topic><topic>Amygdala</topic><topic>Animals</topic><topic>Biochemistry</topic><topic>Biological Techniques</topic><topic>Biosynthesis</topic><topic>Biotechnology</topic><topic>Blood-brain barrier</topic><topic>Blood-Brain Barrier - 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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.</abstract><cop>New York</cop><pub>Springer US</pub><pmid>32030628</pmid><doi>10.1007/s12033-020-00239-x</doi><tpages>10</tpages><orcidid>https://orcid.org/0000-0001-8877-8439</orcidid></addata></record> |
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| ispartof | Molecular biotechnology, 2020-03, Vol.62 (3), p.200-209 |
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| subjects | Administration, Ophthalmic Amygdala Animals Biochemistry Biological Techniques Biosynthesis Biotechnology Blood-brain barrier Blood-Brain Barrier - cytology Blood-Brain Barrier - metabolism Brain Brain stem Cell Biology Chemistry Chemistry and Materials Science Constraining Deoxyribonucleic acid DNA Eye (anatomy) Feasibility studies Flags Gene Expression HEK293 Cells Human Genetics Humans Hydroxylase Male Mice Mice, Inbred BALB C mRNA Nerves Optic Nerve - cytology Optic Nerve - metabolism Original Paper Plasmids - genetics Plasmids - pharmacokinetics Plasmids - pharmacology Protein Science Recombinant Recombinant Fusion Proteins - biosynthesis Recombinant Fusion Proteins - genetics Serotonin Tryptophan Tryptophan hydroxylase Tryptophan Hydroxylase - biosynthesis Tryptophan Hydroxylase - genetics |
| title | Ophthalmic Administration of a DNA Plasmid Harboring the Murine Tph2 Gene: Evidence of Recombinant Tph2-FLAG in Brain Structures |
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