Male Muta™ Mice (CD2-LacZ80/HazfBR) were purchased at 8 weeks of age from Japan Laboratory Animals, Inc. (Tokyo, Japan). Administration of perillaldehyde started at 9 weeks of age. Six mice each were treated with perillaldehyde at 125, 250, 500, or 1000 mg/kg/day by oral gavage for 28 days, with corn oil used as a vehicle. Three days after the final treatment, the liver and glandular stomach were each collected and stored. As a positive control group, mice were treated with N-ethyl-N-nitrosourea (ENU) at 100 mg/kg/day by intraperitoneal (i.p.) injection for two consecutive days. Genomic DNA was extracted from the liver and glandular stomach (whole tissue) using the phenol/chloroform method. Transgenes were rescued via an in vitro packaging reaction using Transpack Packaging Extracts (Agilent Technologies, CA). Mutant frequency (MF) was estimated via the lacZ positive selection method [22 (link)]. Five mice each from the highest three dose groups (i.e., 250, 500, and 1000 mg/kg/day) were used for mutation assays. MFs were statistically analyzed using Dunnett’s test to compare treated groups against the vehicle control group and using Student’s or Welch’s t-test to compare the positive control group against the vehicle control. A significance level of 5% was adopted with two-tailed tests.
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14 protocols using transpack packaging extract
1
Evaluating Perillaldehyde-Induced Mutagenesis
2
Efficient Lambda Phage DNA Packaging
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Packaging efficiency was evaluated as described [19 (link)]. High molecular weight genomic DNA was extracted from the liver of gpt delta rat lines using RecoverEase DNA Isolation Kit (Agilent Technologies, Santa Clara, CA). Lambda EG10 phages were rescued by an in vitro packaging reaction using Transpack Packaging Extracts (Agilent Technologies). For each packaging reaction, 10 µL DNA was employed. E. coli C cells were infected with the packaged samples, mixed with molten soft agar, poured onto lambda agar plates, and incubated at 37 °C overnight. Phage plaques were counted and the number of rescued phages (plaque forming unit: p.f.u.) per packaging reaction was estimated. In addition, E.coli YG6020 cells were infected with the packaged phages, mixed with molten soft agar, poured onto M9 agar plates containing chloramphenicol (Cm), and incubated at 37 °C for 3 days. Cm-resistant colonies were counted and the number of rescued phages (colony forming units: c.f.u.) was estimated.
3
Cisplatin-induced Mutation Frequency Assay
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Forty-eight hours after siRNA knockdown, cells were washed with PBS buffer (GenDEPOT) and treated with 30 µM cisplatin for 1 h. Fresh growth DMEM medium (GenDEPOT) was added and cells were incubated for 24 h. After the 24-h incubation period, the second siRNA transfection was carried out to maintain the siRNA knockdown of the target gene(s). Cells were incubated for an additional 4 d to allow for mutation fixation. Mouse genomic DNA was isolated using the genomic DNA isolation kit (Qiagen). The LIZ shuttle vector was rescued from the genomic DNA by mixing DNA aliquots and transpack packaging extract (Agilent), and the cII assay was carried out as previously described (Yoon et al. 2009 (link), 2010 (link)). The mutation frequency was calculated by dividing the number of mutant plaques by the number of total plaques. For mutation analysis, the sequences of PCR products of the cII gene from the mutant plaques were analyzed as described previously (Yoon et al. 2009 (link), 2010 (link)).
4
Extraction and Phage Recovery from Lung DNA
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High-molecular-weight genomic DNA was extracted from the lungs by using the RecoverEase DNA Isolation Kit (Agilent Technologies, Santa Clara, CA, USA). Lambda EG10 phages containing the gpt gene were recovered from the genomic DNA by using Transpack Packaging Extract (Agilent Technologies).
5
Isolation of 6-TG-Resistant Mutants
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The gpt assay was performed as described previously [31 (link)]. Briefly, DNA was extracted from lung tissue by using a RecoverEase DNA Isolation Kit (Agilent Technologies, Santa Clara, CA, USA) and lambda EG10 phages were rescued by using Transpack Packaging Extract (Agilent Technologies). E. coli YG6020 were infected with the rescued phages, inoculated to M9 salt plates containing chloramphenicol (Cm) and 6-thioguanine (6-TG), and then incubated for 72–90 h at 37 °C, which enabled selection of colonies harboring a plasmid carrying both the gene for chloramphenicol acetyltransferase as well as a mutated gpt gene. Isolates exhibiting the 6-TG-resistant phenotype were cultured overnight at 37 °C in Luria–Bertani (LB) broth containing 25 μg/mL Cm, harvested by centrifugation (7000 rpm, 10 min), and then stored at − 80 °C.
6
Genomic DNA Extraction and Phage Packaging
7
In Vitro Mutagenicity Assay with GPT
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The gpt assay was performed following the method described by Nohmi [29 (link), 32 ]. Our methodology is described below. Briefly, DNA was extracted from the liver, spleen, bone marrow and testis of mice 7 days after IF-MF exposure. These tissues were processed using proteinase K, and the proteins in these solutions were removed via the extraction with phenol, chloroform and isoamyl alcohol (25:24:1). The genomic DNA was purified via ethanol precipitation. The lambda EG10 DNA was rescued in the form of phages using the in vitro packaging reaction with Transpack Packaging Extract (Agilent Technologies, Santa Clara, CA, USA), as per the following protocol. The Lambda EG10 phages were transfected into Escherchia coli YG6020 that expressed Cre-recombinase and generated a 6.4 kb plasmid, which carried the gpt and chloramphenicol acetyltransferase genes. Mutations in the gpt gene were selected using 6-thioguamine (6-TG). We compared the mutant frequency values in these organs between the sham- and IF-MF-exposed groups. The mutant frequency denotes the total number of colonies on the mutation-detection plate (M9 + Chloramphenicol (Cm) + 6-TG) multiplied by 106 and divided by the total number of colonies on the normal-detection plate (M9 + Cm). The ENU-administered group was used as the positive control, whereas the untreated group was used as the negative control.
8
Quantifying gpt Mutation Frequency
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The gpt mutation assay was performed as described previously12 (link)). Briefly, DNA was extracted from
the small intestine mucosa by means of a RecoverEase DNA Isolation Kit (Agilent
Technologies, Santa Clara, CA, USA), and lambda EG10 phages were recovered with Transpack
Packaging Extract (Agilent Technologies). Escherichia coli YG6020 were
infected with the recovered phages, plated on M9 salt plates containing chloramphenicol
(Cm) and 6-thioguanine (6-TG), and then incubated for 72–90 h at 37°C. This incubation
enabled selection of colonies harboring a plasmid carrying both the gene for
chloramphenicol acetyltransferase and a mutated gpt gene.
gpt-Mutant frequency was calculated by dividing the number of mutated
colonies growing on agar plates containing Cm and 6-TG by the number of colonies growing
on agar plates containing Cm alone. The mutants exhibiting the 6-TG-resistant phenotype
were cultured overnight at 37°C in Luria–Bertani broth containing 25 µg/mL Cm, harvested
by centrifugation (7000 rpm, 10 min), and then stored at −80°C. A 739-bp DNA fragment
containing gpt was amplified by means of the polymerase chain reaction
and sequenced as described previously12 (link),13 (link)).
the small intestine mucosa by means of a RecoverEase DNA Isolation Kit (Agilent
Technologies, Santa Clara, CA, USA), and lambda EG10 phages were recovered with Transpack
Packaging Extract (Agilent Technologies). Escherichia coli YG6020 were
infected with the recovered phages, plated on M9 salt plates containing chloramphenicol
(Cm) and 6-thioguanine (6-TG), and then incubated for 72–90 h at 37°C. This incubation
enabled selection of colonies harboring a plasmid carrying both the gene for
chloramphenicol acetyltransferase and a mutated gpt gene.
gpt-Mutant frequency was calculated by dividing the number of mutated
colonies growing on agar plates containing Cm and 6-TG by the number of colonies growing
on agar plates containing Cm alone. The mutants exhibiting the 6-TG-resistant phenotype
were cultured overnight at 37°C in Luria–Bertani broth containing 25 µg/mL Cm, harvested
by centrifugation (7000 rpm, 10 min), and then stored at −80°C. A 739-bp DNA fragment
containing gpt was amplified by means of the polymerase chain reaction
and sequenced as described previously12 (link),13 (link)).
9
Identification of gpt Mutations in Rescued Phages
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Lambda EG10 was rescued from F344 gpt delta rat genomic DNA by in vitro packaging using Transpack Packaging Extract (Agilent technologies, Santa Clara, CA). Rescued phages were infected with E. coli YG6020. Colonies possessing the converted plasmid containing gpt were grown on M9 with 25 μM Cm agar plates for 3 days at 37°C. In total, 30 Cmr clones were randomly picked and sequenced for gpt using an ABI3100 Genetic Analyzer. Two of the clones possessed a T to A transversion at position 299 in gpt. These two gpt mutants and two control clones without mutations in gpt were incubated at 37°C in LB with Cm overnight. The overnight culture was diluted to a concentration of 1 × 100–1 × 108 with LB medium and spotted onto M9 with Cm or M9 with Cm and 25 μM 6TG agar plates. The plates were incubated at 37°C for two days. As positive and negative controls, YG6020 clones harboring gpt− plasmids with a G to A transition at 185 or a GGG to GG deletion at 416–418 and gpt+ plasmid with no mutation in gpt, respectively, were used.
10
Identifying Mutations in gpt Gene
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