Every condition included nearly equal quantities of both male and female Sprague–Dawley rats (Rattus norvegicus; total N = 131) randomly assigned from at least two litters, bred in the University of Nebraska at Omaha Vivarium (see Table 1 for specific conditions). Rats were socially housed in clear Plexiglass cages on a 12:12 light–dark cycle with free access to tap water and food pellets (Teklad). Day of birth was denoted as postnatal day 0 (P0), and rats were weaned at P25. To facilitate comparable nutrition across litters and development, all experimental litters contained between seven and 12 pups through weaning. To both monitor health and collect data on weight changes during development, all rats were weighed daily for the duration of the experiment. All animal procedures were approved by the University of Nebraska at Omaha Institutional Animal Care Committee's and conformed to the guidelines set forth by the National Institutes of Health and the United States National Research Council.
Food pellets
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Food pellets are a type of laboratory equipment designed to provide a consistent and controlled source of nutrition for animals used in research. They are typically composed of a carefully formulated blend of nutrients, grains, and other ingredients to meet the specific dietary requirements of the research subjects.
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9 protocols using food pellets
1
Developmental Neuroscience Protocols in Rats
2
Conditional Inducible Mouse Model
3
Genetically Modified Mice for Colon Cancer
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ES cell-derived mice were produced by blastocyst injection and animals were maintained on a mixed C57B6/129 background. Progeny of both sexes were used for experiments and were genotyped for specific alleles (Lgr5-CreER, Apcflox, R26-rtTA and col1a1) using primers and protocols previously described6 (link)11 (link). Production of mice and all treatments described were approved by the Institutional Animal Care and Use Committee (IACUC) at Weill Cornell Medicine (NY), under protocol number 2014-0038. Doxycycline was administered via food pellets (200 mg kg−1) (Harlan Teklad) at 5–6 weeks of age. Animals were removed from doxycycline diet after 10 days and collected at various time points, detailed in the Results section. For LGK974 treatment studies, LGK974 (5 mg kg−1, Selleckchem #S7143) was mixed with 0.5% methylcellulose and 0.1% Tween 80 and then administrated by daily oral gavage. Animals were weighed everyday during treatment and the mice were killed after 7 days. Animal studies were not blinded during treatment, however, quantitation of tumour burden involved measurements by two parties, one blinded to the treatment groups.
4
Conditional Gene Expression in Transgenic Mice
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Details of the targeting construct, pBS31’-RBGpA TREtight ColA1, are in the Supplementary Information . A DOX-responsive element controlling SRSF6 expression was targeted downstream of the Cola1 locus by Flp/FRT recombinase-mediated site-specific integration in KH2 ES cells, as described50 (link),51 (link). Injection of targeted ES cells into tetraploid blastocysts to produce fully ES-cell-derived transgenic mice was performed by the CSHL Gene Targeting Shared Resource. DOX was administered to adult mice via food pellets (625 mg/kg) (Harlan Teklad). Hair removal was performed either by shaving, plucking, or applying NAIR lotion for 3 min (Church & Dwight Co) immediately before DOX administration. In vivo imaging of skin GFP expression was measured using a Xenogen IVIS imaging system (Perkin Elmer). No experiments were blinded; each group consisted of gender- and age-matched mice (6 to 52 weeks old C57/BL6/mixed background). As the skin phenotype was not anticipated, we did not perform a power calculation to deduce an adequate group size; instead we used a small group size of 8 treated and 8 control animals, which proved adequate for statistical-significance tests. All mouse experiments were approved by the CSHL Animal Care and Use Committee.
5
Sprague Dawley Rat Model Characterization
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Male and female Sprague Dawley rats (n = 72) bred in the University of Nebraska at Omaha Vivarium were used, with date of birth being denoted as P0 (postnatal day 0). A rat model was selected to correspond with our previous work demonstrating differences in recovery and microglia responses between CTX occurring in adult or neonatal rats (Martin et al., 2019 (link); Riquier and Sollars, 2017 (link); Sollars, 2005 (link)). Every condition included both male and female rats randomly assigned from at least two litters. Rats were kept on a 12:12 light-dark cycle in clear Plexiglas cages with free access to tap water and food pellets (Teklad). Rats were weaned at P25 and socially housed. All procedures were carried out in accordance with the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978) and were approved by the local Institutional Animal Care and Use Committee.
6
Conditional Colon Tumor Induction in Mice
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All experiments were performed in accordance with the Memorial Sloan Kettering Institutional Animal Care and Use Committee (IACUC) under protocol number 11‐06‐012. Doxycycline was administered via food pellets (625 mg·kg−1) (Harlan Teklad). 4‐Hydroxytamoxifen (4OHT, Sigma Aldrich, St. Louis, MO, USA, 70% Z‐isomer) was delivered by a single intraperitoneal injection (0.5 mg/mouse) at 5–6 weeks of age. LSL‐Kras (B6.129S4‐Krastm4Tyj/J) and Lgr5‐CreER (B6.129P2‐Lgr5tm1(cre/ERT2)Cle/J) animals were purchased from Jackson Laboratories. CAGs‐LSL‐rtTA3 (B6. Cg‐Gt(ROSA)26Sortm1(CAG‐rtTA3)Slowe/LdowJ) and TG‐Apc.3374 (Col1a1tm4(tetO‐GFP/RNAi:Apc)Slowe) mice were described previously [27 (link), 28 (link)]. We used a GEMM in which the Apc gene can be conditionally suppressed using a doxycycline‐regulated shRNA to develop colon tumors (shApc/Kraswt mice), as described previously [27 (link), 28 (link)]. A KRAS‐mt line was developed by crossing conditional KRAS‐mt allele‐carrying mice (LSL‐KrasG12D) with the shApc mice to develop shApc/KrasG12D mice. Mouse colonic organoids were isolated as previously published [28 (link)].
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Inducible Mouse Models via Blastocyst Injection
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Doxycycline-Inducible Mouse Model
9
Inducible Mouse Models via Blastocyst Injection
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Production of mice and all treatments described were approved by the Institutional Animal Care and Use Committee (IACUC) at Memorial Sloan Kettering Cancer Center (NY), under protocol number 11-06-012. ES cell-derived mice were produced by blastocycst injection by the MSKCC transgenic core facility. Animals were maintained on a mixed C57B6/129 and progeny from breeding were genotyped for specific alleles (R26-rtTA and col1A1) using primers and protocols previously described 17 (link),24 (link). Doxycycline was administered via food pellets (625mg/kg) (Harlan Teklad). Animal studies were not blinded. For detection of p53 protein, animals were sacrificed 4hrs following 6Gy whole body irradiation. For hydrodynamic plasmid delivery 12.5 μ g of pCAGs-rtTA3 plasmid was mixed with sterile 0.9% sodium chloride solution. Mice were injected with a total amount of sodium chloride/plasmid mix corresponding to 10 % of the body weight into the lateral tail vein within 5-7 seconds.
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