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2005 Projects

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Browsing 2005 Projects by Subject "Gene expression"

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    Effects of body condition scores and fat depots on gene expression of fatty acid synthase in horses
    (Colorado State University. Libraries, 2005) Doran, Lynn A., author; Yemm, R., author; Dickinson, C. E., author; Hossner, Kim L., author
    The current study was designed to examine the expression of fatty acid synthase (FAS) genes in horse adipose tissue. Subcutaneous and visceral fat samples were collected from Quarter horses with obese or normal body condition scores and four fat samples were taken from clinical admission horses that were diagnosed with Cushing's disease at the C.S.U. Veterinary Teaching Hospital. Total RNA was isolated from the fat samples and analyzed by ribonuclease protection assay. A nonisotopic ribonucleic probe for fatty acid synthase (FAS) mRNA was constructed from sheep RNA and was utilized for analysis. The results from the ribonuclease protection assays on the four horses with both visceral and subcutaneous fat samples were analyzed to determine the effects of body condition scores and fat depots on the level of adipose tissue FAS mRNA expression. No difference in FAS gene expression was seen in horses with normal (BCS=3) or obese (BCS=5) body condition scores. The expression of FAS in visceral versus subcutaneous fat depots was numerically but not statistically significant. The data suggest that an ovine probe for FAS mRNA can be effectively used in horses. Although no differences were observed between fat depots, body condition score or Cushing's horses, use of larger sample numbers may provide significant results.
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    The effect of over-expression of ζ-crystallin on glutaminase mRNA stability
    (Colorado State University. Libraries, 2005) Propst, Keri J., author; Taylor, Lynn, author; Lee, Yeon, author; Curthoys, Norman P., author
    During metabolic acidosis, increased renal catabolism of glutaminegenerates ammonium and bicarbonate ions to partially restore normal acid-basebalance. The remaining carbons derived from glutamine are then used to synthesizeglucose. This adaptive response is sustained in part by a pH-responsive increase inglutaminase (GA) that results from selective stabilization of the GA mRNA.Previous studies have shown that the 3’-UTR of the GA mRNA contains a pHresponseelement that consists of a direct repeat of an eight-base AU sequence andthat this element binds ζ-crystallin with high affinity and specificity. Increasedbinding of this protein during metabolic acidosis may initiate the pH-responsivestabilization of the GA mRNA. A tetracycline-responsive expression system (tet-off) was developed to test the effect of over-expression of ζ-crystallin on the expression and the stability of the GA mRNA. Two constructs, pcDNA 3.1-βG-GA–Hygro and pTRE2-ζ-crystallin, were created. The pcDNA 3.1/Hygro vector is designed for high-level,constitutive expression in mammalian cell lines and contains the selectable marker,hygromycin. A chimeric βG-GA cDNA segment that encodes β-globin and the 3’-UTR of the GA mRNA was inserted into the pcDNA 3.1/Hygro vector. Theconstruct, pTRE2-ζ-crystallin contains the tet-responsive element (TRE) that drivesthe expression of ζ-crystallin. The two plasmids were co-transfected into 8C cellsthat express high levels of the tTA protein that binds to and activates transcriptionfrom the TRE only in the absence of doxycycline (Dox). Clonal cell lines wereselected with hygromycin. These cells were grown in the presence and absence ofDox and screened with ζ-crystallin specific antibodies to identify clonal lines thatexhibit a large induction of ζ-crystallin when grown in the absence of Dox. RNAisolated from the selected line was quantified using Real-Time RT-PCR. Theresulting data demonstrate that over-expression of ζ-crystallin does not increase GAmRNA levels.