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Zinc-finger nuclease knockout of dual-specificity protein phosphatase-5 enhances the myogenic response and autoregulation of cerebral blood flow in FHH.1BN rats.

Fan F, Geurts AM, Pabbidi MR, Smith SV, Harder DR, Jacob H, Roman RJ - PLoS ONE (2014)

Bottom Line: We recently reported that the myogenic responses of the renal afferent arteriole (Af-Art) and middle cerebral artery (MCA) and autoregulation of renal and cerebral blood flow (RBF and CBF) were impaired in Fawn Hooded hypertensive (FHH) rats and were restored in a FHH.1BN congenic strain in which a small segment of chromosome 1 from the Brown Norway (BN) containing 15 genes including dual-specificity protein phosphatase-5 (Dusp5) were transferred into the FHH genetic background.The expression of Dusp5 was higher at the mRNA level but not at the protein level and the levels of p-ERK1/2 and p-PKC were lower in cerebral microvessels and brain tissue isolated from FHH than in FHH.1BN rats.These results indicate that Dusp5 modulates myogenic reactivity in the cerebral circulation and support the view that a mutation in Dusp5 may enhance Dusp5 activity and contribute to the impaired myogenic response in FHH rats.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, Mississippi, United States of America.

ABSTRACT
We recently reported that the myogenic responses of the renal afferent arteriole (Af-Art) and middle cerebral artery (MCA) and autoregulation of renal and cerebral blood flow (RBF and CBF) were impaired in Fawn Hooded hypertensive (FHH) rats and were restored in a FHH.1BN congenic strain in which a small segment of chromosome 1 from the Brown Norway (BN) containing 15 genes including dual-specificity protein phosphatase-5 (Dusp5) were transferred into the FHH genetic background. We identified 4 single nucleotide polymorphisms in the Dusp5 gene in FHH as compared with BN rats, two of which altered CpG sites and another that caused a G155R mutation. To determine whether Dusp5 contributes to the impaired myogenic response in FHH rats, we created a Dusp5 knockout (KO) rat in the FHH.1BN genetic background using a zinc-finger nuclease that introduced an 11 bp frame-shift deletion and a premature stop codon at AA121. The expression of Dusp5 was decreased and the levels of its substrates, phosphorylated ERK1/2 (p-ERK1/2), were enhanced in the KO rats. The diameter of the MCA decreased to a greater extent in Dusp5 KO rats than in FHH.1BN and FHH rats when the perfusion pressure was increased from 40 to 140 mmHg. CBF increased markedly in FHH rats when MAP was increased from 100 to 160 mmHg, and CBF was better autoregulated in the Dusp5 KO and FHH.1BN rats. The expression of Dusp5 was higher at the mRNA level but not at the protein level and the levels of p-ERK1/2 and p-PKC were lower in cerebral microvessels and brain tissue isolated from FHH than in FHH.1BN rats. These results indicate that Dusp5 modulates myogenic reactivity in the cerebral circulation and support the view that a mutation in Dusp5 may enhance Dusp5 activity and contribute to the impaired myogenic response in FHH rats.

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Schematic describing the generation of Dusp5 ZFN KO rats in the FHH.1BN genetic background.Panel A presents the sequence of the Dusp5 ZFN. The Dusp5 specific ZFN introduced a 14 bp deletion and a 3 bp insertion resulting in a net 11 bp deletion between nucleotides 449–464 in the Dusp5 sequence in the KO animals that introduced a frame shift mutation. Panel B presents the strategy for the generation of Dusp5 ZFN KO rats in the FHH.1BN genetic background. Fertilized donor embryos from female FHH.1BN rats were collected and the Dusp5 ZFN mRNA was microinjected into pronuclei of the fertilized one-cell embryos. These embryos were transferred back to a foster mother. The heterozygous founders were brother-sister mated to generate the homozygous ZFN KO founders and the FHH.1BN wild type control rats. Panel C presents an example of PCR genotyping of the region of interest in FHH.1BN and Dusp5 KO rats. The rats were genotyped using the following primers: Dusp5-F: 5′-GCT GCA GGA GGG CGG CGG CG -3′, Dusp5 R: 5′-CTT TAA GGA AGT AGA CCC G-3′. These primers amplify a 155 bp band for the wild type allele and a 144 bp band for the knockout allele. Both bands are observed in heterozygous rats.
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pone-0112878-g004: Schematic describing the generation of Dusp5 ZFN KO rats in the FHH.1BN genetic background.Panel A presents the sequence of the Dusp5 ZFN. The Dusp5 specific ZFN introduced a 14 bp deletion and a 3 bp insertion resulting in a net 11 bp deletion between nucleotides 449–464 in the Dusp5 sequence in the KO animals that introduced a frame shift mutation. Panel B presents the strategy for the generation of Dusp5 ZFN KO rats in the FHH.1BN genetic background. Fertilized donor embryos from female FHH.1BN rats were collected and the Dusp5 ZFN mRNA was microinjected into pronuclei of the fertilized one-cell embryos. These embryos were transferred back to a foster mother. The heterozygous founders were brother-sister mated to generate the homozygous ZFN KO founders and the FHH.1BN wild type control rats. Panel C presents an example of PCR genotyping of the region of interest in FHH.1BN and Dusp5 KO rats. The rats were genotyped using the following primers: Dusp5-F: 5′-GCT GCA GGA GGG CGG CGG CG -3′, Dusp5 R: 5′-CTT TAA GGA AGT AGA CCC G-3′. These primers amplify a 155 bp band for the wild type allele and a 144 bp band for the knockout allele. Both bands are observed in heterozygous rats.

Mentions: The homozygous Dusp5 knockout (KO) and wild type FHH.1BN control strain were derived from an intercross of the heterozygous Dusp5 founders. Genotyping and sequencing of the Dusp5 KO strain indicated that there is a 14 bp deletion and a 3 bp insertion between nucleotides 449–464 in Dusp5 mRNA that creates a frame shift mutation which is predicted to introduce a premature stop codon at amino acid (AA) 121 (Figure 2A,4A, 4B). The genotypes of the animals were verified using PCR as shown in Figure 4C.


Zinc-finger nuclease knockout of dual-specificity protein phosphatase-5 enhances the myogenic response and autoregulation of cerebral blood flow in FHH.1BN rats.

Fan F, Geurts AM, Pabbidi MR, Smith SV, Harder DR, Jacob H, Roman RJ - PLoS ONE (2014)

Schematic describing the generation of Dusp5 ZFN KO rats in the FHH.1BN genetic background.Panel A presents the sequence of the Dusp5 ZFN. The Dusp5 specific ZFN introduced a 14 bp deletion and a 3 bp insertion resulting in a net 11 bp deletion between nucleotides 449–464 in the Dusp5 sequence in the KO animals that introduced a frame shift mutation. Panel B presents the strategy for the generation of Dusp5 ZFN KO rats in the FHH.1BN genetic background. Fertilized donor embryos from female FHH.1BN rats were collected and the Dusp5 ZFN mRNA was microinjected into pronuclei of the fertilized one-cell embryos. These embryos were transferred back to a foster mother. The heterozygous founders were brother-sister mated to generate the homozygous ZFN KO founders and the FHH.1BN wild type control rats. Panel C presents an example of PCR genotyping of the region of interest in FHH.1BN and Dusp5 KO rats. The rats were genotyped using the following primers: Dusp5-F: 5′-GCT GCA GGA GGG CGG CGG CG -3′, Dusp5 R: 5′-CTT TAA GGA AGT AGA CCC G-3′. These primers amplify a 155 bp band for the wild type allele and a 144 bp band for the knockout allele. Both bands are observed in heterozygous rats.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4232417&req=5

pone-0112878-g004: Schematic describing the generation of Dusp5 ZFN KO rats in the FHH.1BN genetic background.Panel A presents the sequence of the Dusp5 ZFN. The Dusp5 specific ZFN introduced a 14 bp deletion and a 3 bp insertion resulting in a net 11 bp deletion between nucleotides 449–464 in the Dusp5 sequence in the KO animals that introduced a frame shift mutation. Panel B presents the strategy for the generation of Dusp5 ZFN KO rats in the FHH.1BN genetic background. Fertilized donor embryos from female FHH.1BN rats were collected and the Dusp5 ZFN mRNA was microinjected into pronuclei of the fertilized one-cell embryos. These embryos were transferred back to a foster mother. The heterozygous founders were brother-sister mated to generate the homozygous ZFN KO founders and the FHH.1BN wild type control rats. Panel C presents an example of PCR genotyping of the region of interest in FHH.1BN and Dusp5 KO rats. The rats were genotyped using the following primers: Dusp5-F: 5′-GCT GCA GGA GGG CGG CGG CG -3′, Dusp5 R: 5′-CTT TAA GGA AGT AGA CCC G-3′. These primers amplify a 155 bp band for the wild type allele and a 144 bp band for the knockout allele. Both bands are observed in heterozygous rats.
Mentions: The homozygous Dusp5 knockout (KO) and wild type FHH.1BN control strain were derived from an intercross of the heterozygous Dusp5 founders. Genotyping and sequencing of the Dusp5 KO strain indicated that there is a 14 bp deletion and a 3 bp insertion between nucleotides 449–464 in Dusp5 mRNA that creates a frame shift mutation which is predicted to introduce a premature stop codon at amino acid (AA) 121 (Figure 2A,4A, 4B). The genotypes of the animals were verified using PCR as shown in Figure 4C.

Bottom Line: We recently reported that the myogenic responses of the renal afferent arteriole (Af-Art) and middle cerebral artery (MCA) and autoregulation of renal and cerebral blood flow (RBF and CBF) were impaired in Fawn Hooded hypertensive (FHH) rats and were restored in a FHH.1BN congenic strain in which a small segment of chromosome 1 from the Brown Norway (BN) containing 15 genes including dual-specificity protein phosphatase-5 (Dusp5) were transferred into the FHH genetic background.The expression of Dusp5 was higher at the mRNA level but not at the protein level and the levels of p-ERK1/2 and p-PKC were lower in cerebral microvessels and brain tissue isolated from FHH than in FHH.1BN rats.These results indicate that Dusp5 modulates myogenic reactivity in the cerebral circulation and support the view that a mutation in Dusp5 may enhance Dusp5 activity and contribute to the impaired myogenic response in FHH rats.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, Mississippi, United States of America.

ABSTRACT
We recently reported that the myogenic responses of the renal afferent arteriole (Af-Art) and middle cerebral artery (MCA) and autoregulation of renal and cerebral blood flow (RBF and CBF) were impaired in Fawn Hooded hypertensive (FHH) rats and were restored in a FHH.1BN congenic strain in which a small segment of chromosome 1 from the Brown Norway (BN) containing 15 genes including dual-specificity protein phosphatase-5 (Dusp5) were transferred into the FHH genetic background. We identified 4 single nucleotide polymorphisms in the Dusp5 gene in FHH as compared with BN rats, two of which altered CpG sites and another that caused a G155R mutation. To determine whether Dusp5 contributes to the impaired myogenic response in FHH rats, we created a Dusp5 knockout (KO) rat in the FHH.1BN genetic background using a zinc-finger nuclease that introduced an 11 bp frame-shift deletion and a premature stop codon at AA121. The expression of Dusp5 was decreased and the levels of its substrates, phosphorylated ERK1/2 (p-ERK1/2), were enhanced in the KO rats. The diameter of the MCA decreased to a greater extent in Dusp5 KO rats than in FHH.1BN and FHH rats when the perfusion pressure was increased from 40 to 140 mmHg. CBF increased markedly in FHH rats when MAP was increased from 100 to 160 mmHg, and CBF was better autoregulated in the Dusp5 KO and FHH.1BN rats. The expression of Dusp5 was higher at the mRNA level but not at the protein level and the levels of p-ERK1/2 and p-PKC were lower in cerebral microvessels and brain tissue isolated from FHH than in FHH.1BN rats. These results indicate that Dusp5 modulates myogenic reactivity in the cerebral circulation and support the view that a mutation in Dusp5 may enhance Dusp5 activity and contribute to the impaired myogenic response in FHH rats.

Show MeSH
Related in: MedlinePlus