F these Terc gene cluster variants on absolute liver telomere length in an independent panel of inbred mouse strains chosen according to genotype at candidate SNPs within the chromosome three cluster. This second experiment supported our finding that polymorphisms within the Terc gene cluster influence telomere length in inbred mouse strains, replicating findings in human populations. These findings provide support for inbred mouse strains as a model for telomere dynamics, specifically for studying mechanisms underlying the association between Terc gene cluster variants and telomere length. 2. Materials and Approaches two.1. Experiment 1 2.1.1. Experiment 1: Overview The initial aim of Experiment 1 was to test effects of 2-NBDG Epigenetic Reader Domain nicotine exposure on liver telomere length within a panel of inbred mouse strains. Animals were a a part of a larger project testing effects of nicotine exposure and genetic background on fear conditioning. Therefore, animals were previously exposed to a cued/contextual fear conditioning paradigm (ending 1 day prior to euthanasia). Subjects had been also exposed to 18 mg/kg/day nicotine or saline more than a period of 12 days via subcutaneous osmotic minipump. Liver tissue for telomere length quantification was dissected three days following removal of drug or car. Worry conditioning and drug exposure methodology can be located in Supplementary Supplies. two.1.2. Experiment 1: Subjects The subjects were adult (103 weeks at time of liver dissection), male mice of eight inbred mouse strains: 129S2/SvPasCrl, 129S4/SvJaeJ, 129S8/SvEvNimrJ, BTBR T+ Itpr3tf/J, C57BL/6J, MA/MyJ, NZB/BINJ and SM/J (n = 9 per therapy group per strain, all strains apart from 129S2/SvPasCrl bought from Jackson Laboratory, Bar Harbor, ME, USA; 129S2/SvPasCrl purchased from Charles River, Wilmington, MA, USA). All mice were group-housed in the same colony room using a 12 h light/dark cycle and ad libitum access to food and water. All procedures were performed in accordance using the NIH Guide forCells 2021, ten,four ofthe Care and Use of Laboratory Animals and were approved by the Pennsylvania State University IACUC committee. 2.1.three. Experiment 1: Liver Dissection and DNA Extraction Liver tissue from the left lobe was dissected immediately following euthanasia by cervical dislocation, which occurred 3 days soon after osmotic minipump removal. Dissections were performed at room temperature and dissected tissue was stored at -80 C. DNA was extracted from liver tissue making use of the Qiagen DNeasy Blood and Tissue Kit (Hilden, Germany) based on the manufacturer’s instructions. DNA purity was assessed utilizing 260/280 and 260/230 absorbance ratio readings on YB-0158 manufacturer NanoDrop 2000 (Thermo Scientific; Wilmington, DE, USA). Liver DNA concentration was quantified employing the QuantiT PicoGreen dsDNA assay kit (ThermoFisher; Waltham, MA, USA). For Experiment 1, Picogreen DNA quantification was performed by the Penn State Biomarker Core Laboratory. Samples were read around the Synergy two Multi-Mode Plate Reader (Biotek; Winooski, VT, USA) at an excitation wavelength of 485 nm and an emission wavelength of 528 nm. All samples had been diluted to a concentration of 1 ng/ for subsequent telomere length measurement. two.1.four. Experiment 1: Telomere Length Quantification Absolute telomere length (aTL) was measured making use of a quantitative PCR system adapted from O’Callaghan and Fenech [27] (originally adapted from T/S ratio process by Cawthon [28]). Briefly, this assay utilizes an oligomer telomere standard ladder alongside quantific.
