G-ARKO and E-ARKO male mice were generated as previously described (17 (link), 20 (link)). T-ARKO and E-ARKO male mice were generated by breeding AR+/flox female mice with male pLCK-Cre+ mice (Stock no. 003802, B6.Cg-Tg(LCK-Cre)548Jxm/J, Jackson laboratory, Bar Harbor, Maine, USA), and K5-Cre+ mice (19 (link)), respectively. Because our initial assessments of androgen status (wet weight of androgen sensitive organs) and thymus weight and cellularity revealed no differences between AR+ and ARflox males, Cre+ littermates without the ARflox construct were used as controls for subsequent experiments. In all experiments the different ARKO mice were compared to littermate controls and the mice were on a C57BL/6 ApoE constitutive knockout background (B6.129P2-Apoetm1UncN11, Taconic). We assessed AR, Cre, and Zfy (for gender) by PCR amplification of genomic DNA (gDNA) (27 (link)). The mice were housed in a temperature- and humidity-controlled room with a 06:00–18:00 h light cycle and consumed a soy-free diet (R70, Lantmännen) and tap water ad libitum. All animal studies were conducted in compliance with local guidelines and The Ethics Committee on Animal Care and Use in Gothenburg approved all procedures.
B6.129p2 apoetm1uncn11
Manufactured by Taconic Biosciences
Sourced in Denmark
The B6.129P2-Apoetm1UncN11 is a laboratory mouse strain that serves as a model for studying apolipoprotein E (ApoE) deficiency. This strain carries a targeted mutation in the ApoE gene, leading to the production of a dysfunctional form of the ApoE protein. The core function of this product is to provide a standardized, genetically-modified animal model for researchers to investigate the role of ApoE in various biological processes and disease states.
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8 protocols using b6.129p2 apoetm1uncn11
1
Generation and Characterization of ARKO Mice
2
Cigarette Smoke Exposure in ApoE-/- Mice
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All procedures involving animals were performed in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) and licensed by the Singapore National Parks/Animal and Veterinary Service (AVS), with approval from an Institutional Animal Care and Use Committee and in compliance with the National Advisory Committee for Laboratory Animal Research Guidelines on the Care and Use of Animals for Scientific Purposes (NACLAR 2004). Female Apoe−/− mice (B6.129P2-ApoEtm1/Unc N11) bred under specific pathogen-free conditions were obtained from Taconic Biosciences (Rensselaer, NY, USA).
The mice were whole-body exposed to diluted mainstream smoke from 3R4F cigarettes (target concentration: 560 µg TPM/L, equivalent to 35.9 µg nicotine/L). This nicotine concentration was matched in the base and test aerosols, and this base and test TPM concentration was matched in the carrier and test aerosols (Online Resource 1a). Intermittent exposures to fresh filtered air for 30 and 60 min after the first and second hours of exposure, respectively, were provided to avoid buildup of excessive carboxyhemoglobin in the 3R4F CS-exposed mice (Online Resource 1b).
The mice were whole-body exposed to diluted mainstream smoke from 3R4F cigarettes (target concentration: 560 µg TPM/L, equivalent to 35.9 µg nicotine/L). This nicotine concentration was matched in the base and test aerosols, and this base and test TPM concentration was matched in the carrier and test aerosols (Online Resource 1a). Intermittent exposures to fresh filtered air for 30 and 60 min after the first and second hours of exposure, respectively, were provided to avoid buildup of excessive carboxyhemoglobin in the 3R4F CS-exposed mice (Online Resource 1b).
3
ApoE Knockout Mouse Atherosclerosis Study
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Seventy-two pathogen-free female ApoE−/−-mice (B6.129P2-Apoetm1 Unc N11) were purchased from Taconic (Taconic M&B, Ry, Denmark). After arrival at the local animal facility all mice were earmarked and randomly allocated into 6 groups (n = 12) with equal numbers of cages per intervention (n = 4 cages/diet). Due to weight loss during the one-week acclimatization, two mice were excluded (n = 11 in the RWO-I and CWO groups). At the start of the experiment, the mice were 6 weeks of age with a body weight range of 16 to 21 g. Mice provided with water and pelleted feed ad libitum for 13 weeks were kept in ventilated cages placed in the same room in a conventional laboratory animal unit. The temperature and relative humidity were 21 °C and 55% on a 12-h day/night cycle (light: 0600 to 1800 h). The cages and bedding were changed once a week. At the end of the study all mice were feed-deprived for 3 h prior to euthanizing by carbon dioxide inhalation. Blood was drawn by cardiac puncture and serum was prepared and frozen at − 80 °C. Cardiac, hepatic, renal, splenic and adipose tissues were dissected out, weighed, snap frozen and stored at − 80 °C.
4
Atherosclerosis Induction in ApoE-KO Mice
5
Cigarette Smoke Exposure in ApoE-/- Mice
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All procedures involving animals were performed in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care and licensed by the Agri-Food & Veterinary Authority of Singapore, with approval from an Institutional Animal Care and Use Committee (IACUC protocol #15044) and in compliance with the National Advisory Committee for Laboratory Animal Research's Guidelines on the Care and Use of Animals for Scientific Purposes (NACLAR, 2004) . Female ApoE Ϫ/Ϫ mice (B6.129P2-Apo-E tm1/Unc N11), bred under specific pathogen-free conditions, were obtained from Taconic Biosciences (Rensselaer, NY). The health status of the animals was verified by using the health-check certificate provided by the breeder. The mice were maintained and exposed under specific hygienic conditions with filtered conditioned fresh air at 22 Ϯ 2°C and 55 Ϯ 15% humidity. Additional details of animal housing, randomization, and acclimatization have been previously published (13, (link)91, (link)122, (link)123) (link).
The mice were whole body exposed to diluted mainstream smoke from 3R4F cigarettes (achieved concentration, 574.5 g TPM/L, equivalent to 35.9 g nicotine/L), base aerosol (nicotine-matched to 3R4F, 36.4 g nicotine/L; PG-matched to carrier, 171 (link)
The mice were whole body exposed to diluted mainstream smoke from 3R4F cigarettes (achieved concentration, 574.5 g TPM/L, equivalent to 35.9 g nicotine/L), base aerosol (nicotine-matched to 3R4F, 36.4 g nicotine/L; PG-matched to carrier, 171 (link)
6
Anti-CD3 Antibody Immunomodulation in apoE-/- Mice
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At 4 weeks of age, male apoE-deficient mice (B6.129P2-Apoetm1UncN11; Taconic) were bilaterally castrated or sham operated under isoflurane anesthesia. One week later, the mice were injected intraperitoneally with 50 µg anti-mouse CD3 antibody (clone 145-2C11 f[ab′]2 fragments, Cat No. BE0001-1FAB; BioXCell) or a control antibody (hamster IgG f[ab′]2 fragments, Cat No. BE0091-FAB; BioXCell) on 5 consecutive days. As described previously,21 (link) the antibody injections were repeated with 3-week intervals (when the mice were 5, 8, 11, and 14 weeks of age), and the mice were euthanized at 16 weeks of age.
7
Atherosclerosis in Apoe KO Mice
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Male Apoe knockout (Apoe KO) mice B6.129P2-Apoetm1Unc N11 were obtained from Taconic (Borup, Denmark). The mouse model was originally developed through embryonic transfer51 (link) and the line is maintained by inbreeding homozygous mice. As control mice, male wild type (WT) C57BL/6JBomTac mice were used with same genetic background (C57BL/6) as the Apoe KO mice. The animals (WT N = 48, Apoe KO N = 48) were purchased at 10 weeks of age, group housed, and kept on a 12-h light/dark cycle at constant room temperature. Both WT and Apoe KO mice were feed ad libitum water and standard rodent chow (#131003, Altromin, Lage, Germany).
8
ApoE/COMP Knockout Mouse Model for Atherosclerosis
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All animal experiments were approved by the Malmoe/Lund regional ethical committee (Sweden). ApoE-null mice on a BL/6 background (B6.129P2-Apoetm1Unc/N11) purchased from Taconic (Lille Skensved, Denmark) and COMP knockout mice generated by targeted disruption [16] (link) were crossed. From these resulting double heterozygous animals, new strains of ApoE/COMP double-null and ApoE-null mice were generated and female offspring were used in the experiments. At 16 weeks of age, mice were switched from normal chow diet to a high-fat diet containing 21% fat and 0.15% cholesterol (Special Diet Services, UK). Two weeks later surgery was performed, and the high-fat diet was continued for a further 12 weeks. At the age of 30 weeks mice were sacrificed and blood was taken for plasma cytokine and cholesterol analysis, which revealed no significant differences between the double and single knockout mice (Suppl. Tab. 1 ).
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