| Autor: |
Hastings, Nicholas, Xiaohai Wang, Lixin Song, Butts, Brent, Grotz, Diane, Hargreaves, Richard, J. Fred Hess, Kwok-Lam Hong, Huang, Cathy, Hyde, Lynn, Laverty, Maureen, Lee, Julie, Levitan, Diane, Lu, Sherry, Maguire, Maureen, Veeravan Mahadomrongkul, McEachern, Ernest, Xuesong Ouyang, Rosahl, Thomas, Selnick, Harold, Stanton, Michaela, Terracina, Giuseppe, Vocadlo, David, Ganfeng Wang, Duffy, Joseph, Parker, Eric, Zhang, Lili |
| Rok vydání: |
2017 |
| DOI: |
10.6084/m9.figshare.c.3783323_d2 |
| Popis: |
Analysis of OGA mRNA expression in OGA iKD and littermate WT mice after treatment for 10 days with or without doxycycline. shRNA-mediated knock-down of OGA was achieved by in-diet treatment with doxycycline for 10 days as described in Additional file 1, after which animals (n = 3 per group) were sacrificed and various tissues were harvested. The RNeasy Lipid Tissue Mini Kit (Qiagen) was used for RNA isolation from tissues according to the manufacturer’s instructions. Pre-configured TaqMan Gene Expression Assays (Applied Biosystems) were used to assess the OGA mRNA level with the Hp1bp3 housekeeping gene as the normalization control. To compare OGA mRNA expression levels between treatment and genotype, the CT values for WT mice without doxycycline treatment were set at 100% and CT values for the other treatment/genotype groups were expressed relative to that value. Absolute CT values for each tissue were: brain, 24; aorta, 26; heart, 25; muscle, 25; SI, 25; colon, 24; EWAT, 26; kidney, 23; liver, 25; pancreas, 30. OGA mRNA expression was detected in all tissues examined, with particularly high expression levels in brain, colon, and kidney. About 70% knock-down of OGA mRNA levels was achieved in brain in the OGA iKD mice treated with doxycycline, but a 35% knock-down of OGA mRNA was also noted for OGA iKD mice that were fed diet without doxycycline, indicating some leakiness in the expression of the shRNA construct in the brain. No other tissue showed evidence of leaky expression of the shRNA construct in the absence of doxycycline treatment. Substantial knock-down of OGA mRNA expression was observed in all tissues in response to doxycycline treatment, with the lowest amount of OGA mRNA remaining in the heart (12%), kidney (18%) and liver (25%). Abbreviations: SI, small intestine; EWAT, epididymal white adipose tissue. (PPTX 99 kb) |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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