Abstract 39: Cyp4a1 Transgenic Rats Generated Using Sleeping Beauty Transposon System Restores the Impaired Myogenic Responses in the Afferent Arteriole of Dahl S Rats

We have reported that the production of 20-HETE is reduced in the renal vasculature of Dahl S rats and that myogenic and TGF responses of afferent arteries (Af-Art) are impaired in Dahl S rats. In this study we generated CYP4A1 transgenic rats in the Dahl S inbred strain background utilizing the enhanced Sleeping Beauty (SB100X) transposon system to determine if upregulation of 20-HETE production can restore vascular reactivity and oppose the development of renal injury. Fertilized eggs collected from female Dahl S rats were microinjected with a transposon vector harboring the rat CYP4A1 cDNA under the control of the ubiquitous CAG promoter along with SB100X transposase mRNA to produce transgenic founders. Heterozygous founders were backcrossed to Dahl S rats, transgene insertion sites were identified by Ligation Mediated PCR and sequencing, and the progeny were brother-sister mated to derive homozygous transgenic lines. The expression of CYP4A protein was significantly elevated and the production of 20-HETE was 3-fold higher in the renal outer medullary tissue of CYP4A1 transgenic (n=17) compared to Dahl S rats (n=17). 20-HETE production was 10-fold higher in renal microvessels of CYP4A1 transgenic animals than Dahl S rats. (0.2±0.3, n=22 versus 1.9±0.1 pmol/mg/min, n=14). The luminal diameter of the Af-Art decreased significantly from 15.9 ± 0.6 to 14.1 ± 0.5 μm in CYP4A1 transgenic rats (n=5) when the perfusion pressure was increased from 60 to 120 mmHg, whereas it remained unaltered in Dahl S rats (from 19.4 ± 2.3 to 20.6 ± 5.6 μm, n=22). These studies further support the view that a deficiency in the formation of 20-HETE in the renal microcirculation contributes to the marked susceptibility of Dahl S rats to develop of hypertension and diabetic induced renal injury, and the new CYP4A1 transposon transgenic rat model may be useful for determining the mechanisms involved.

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Abstract 36: Deficiency in the Production of 20-HETE Contributes to Impaired Myogenic and TGF Responses in the Afferent Arteriole of Dahl S Rats

Hypertension ◽

10.1161/hyp.60.suppl_1.a36 ◽

2012 ◽

Vol 60 (suppl_1) ◽

Author(s):

Ying Ge ◽

Fan Fan ◽

Sydney R Murphy ◽

Jan Michael Williams ◽

Ruisheng Liu ◽

...

Keyword(s):

Renal Injury ◽

Tubuloglomerular Feedback ◽

Brown Norway ◽

Sodium Reabsorption ◽

Thick Ascending Limb ◽

Afferent Arteriole ◽

Synthesis Inhibitor ◽

Renal Vasculature ◽

Luminal Diameter ◽

Consomic Strain

Previous studies have indicated that a deficiency in the formation of 20-HETE in the proximal tubule and thick ascending limb of Henle in Dahl S rats increases sodium reabsorption and contributes to the development of hypertension. The present study examined whether the lack of 20-HETE production in the renal vasculature contributes to the progression of renal injury by altering the myogenic or tubuloglomerular feedback (TGF) response of the afferent arteriole (Af-Art). The production of 20-HETE was significantly lower by 54% in renal microvessels isolated from the kidneys of Dahl S rats versus that seen than in SS.5BN consomic strain in which chromosome 5 from the Brown Norway (BN) rat containing the CYP4A genes responsible for the formation of 20-HETE was transferred into the Dahl S genetic background. The luminal diameter of the Af-Art decreased by 14.7± 1.5% (from 20.5 ± 0.7 to 17.5 ± 0.8 μm, n=6) in SS.5BN rats whereas the diameter of the Af-Art remained unaltered in Dahl S rats (from 20.1 ± 0.6 to 21.7 ± 0.6 μm, n=7) when the perfusion pressure was increased from 60 mmHg to 120 mmHg. In other experiments, adenosine (1 μM) reduced the diameter of the Af-Art in the SS.5BN rats by 15±0.7% (from 20.1 ±0.4 to 17.1 ± 0.9 μm, n=3) whereas the Af-Art of Dahl S rats was unaltered. However, administration of a 20-HETE synthesis inhibitor, HET0016 (1 μM, n=6), or a selective 20-HETE antagonist, 6, 15-20-HEDE (10 μM, n=6) completely blocked the myogenic and adenosine responses in the Af-Art of SS.5BN rats but it had no effect in Dahl S rats. Administration of a 20-HETE agonist, 5, 14-20-HEDE (1 μM) restored the myogenic response (from 20.7 ± 0.7 to 17.6 ± 0.6 μm, n=7) and vasoconstrictor response to adenosine in the Af-Art of Dahl S rats. These studies confirm the key role of 20-HETE in modulating the responsiveness of the Af-Art and indicate that a deficiency in the formation of 20-HETE in renal microvessels contributes to the marked susceptibility of Dahl S rats to develop hypertension induced renal injury.

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Generation of CYP4A1 transgenic rats using the Sleeping Beauty transposon system

The FASEB Journal ◽

10.1096/fasebj.25.1_supplement.1099.2 ◽

2011 ◽

Vol 25 (S1) ◽

Author(s):

Fan Fan ◽

Howard Jacob ◽

Aron Geurts ◽

Sydney Murphy ◽

Richard J. Roman

Keyword(s):

Sleeping Beauty ◽

Transgenic Rats ◽

Sleeping Beauty Transposon

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Faculty Opinions recommendation of Mammalian mutagenesis using a highly mobile somatic Sleeping Beauty transposon system.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1026892.329485 ◽

2005 ◽

Author(s):

Donald Rio

Keyword(s):

Sleeping Beauty ◽

Sleeping Beauty Transposon

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Pharmaceutical and Biotechnological Applications of Transgenic Technology

International Journal of Pharmaceutical Sciences and Nanotechnology ◽

10.37285/ijpsn.2018.11.4.1 ◽

2018 ◽

Vol 11 (4) ◽

pp. 4145-4153

Author(s):

D Samba Reddy ◽

Tina Reddy

Keyword(s):

Mammalian Species ◽

Genetic Diseases ◽

Transgenic Animals ◽

Transgenic Animal ◽

Pharmaceutical Products ◽

Conditional Knockout ◽

Knock Out ◽

Transgenic Lines ◽

Health And Disease ◽

Or Genes

A transgenic animal is a genetically modified species in which researchers have modified an existing gene or genes by genetic engineering techniques. Genetic modification involves the mutation, insertion, or deletion of genes. Mouse is the most widely used mammalian species for creating transgenic lines. There are two types of transgenic animals: (i) gene deleted (“knock-out”) and (ii) gene overexpressed (“knock-in”). The loss or gain of gene activity often causes changes in a mouse's phenotype, which includes appearance, behavior and other observable characteristics. Knockout mice are key animal models for studying the role of genes which have been sequenced but whose functions have not been determined. They include constitutive knockouts (gene deleted since birth) and conditional knockout (gene turned off later after birth). The first knockout mouse was created in 1989 by Mario Capecchi, Martin Evans, and Oliver Smithies, for which they were awarded the 2007 Nobel Prize in Physiology or Medicine. Transgenic mouse models have revolutionized the biomedical research and provided a power tool for understanding health and disease. Transgenic animals have been created for bulk production of biotechnology and pharmaceutical products. In 2009, the FDA approved the first human biological drug ATryn, an anticoagulant extracted from the transgenic goat's milk. The recently discovered CRISPER gene editing technology is providing new frontiers in correcting abnormal genes and hopefully provide cures for genetic diseases in the future.

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