Establishment of an Efficient BAC Transgenesis Protocol and its Application to Functional Characterization of the Mouse Brachyury Locus

Abe, Kuniya; Hazama, Masatoshi; Katoh, Hideki; Yamamura, Ken Ichi; Suzuki, Misao

doi:10.1538/expanim.53.311

Cited by 29 publications

(18 citation statements)

References 23 publications

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“…The ϳ210-kbp BAC DNA insert (Fig. 1A) was linearized with NotI, which removes the vector, and was isolated after pulsedfield gel electrophoresis and ␤-agarase digestion, according to a published method (Abe et al, 2004). Transgenic mice were produced at the Transgenic and Knockout Core Facility at the Wadsworth Center (Albany, NY), according to standard procedures (Nagy et al, 2003).…”

Section: Methodsmentioning

confidence: 99%

Generation and Characterization of aCYP2A13/2B6/2F1-Transgenic Mouse Model

Wei

Lei

et al. 2012

Drug Metab Dispos

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ABSTRACT:CYP2A13, CYP2B6, and CYP2F1, which are encoded by neighboring cytochrome P450 genes on human chromosome 19, are active in the metabolic activation of many drugs, respiratory toxicants, and chemical carcinogens. To facilitate studies on the regulation and function of these human genes, we have generated a CYP2A13/2B6/2F1-transgenic (TG) mouse model (all *1 alleles). Homozygous transgenic mice are normal with respect to gross morphological features, development, and fertility. The tissue distribution of transgenic mRNA expression agreed well with the known respiratory tract-selective expression of CYP2A13 and CYP2F1 and hepatic expression of CYP2B6 in humans. CYP2A13 protein was detected through immunoblot analyses in the nasal mucosa (NM) (ϳ100 pmol/mg of microsomal protein; similar to the level of mouse CYP2A5) and the lung (ϳ0.2 pmol/mg of microsomal protein) but not in the liver of the TG mice. CYP2F1 protein, which could not be separated from mouse CYP2F2 in immunoblot analyses, was readily detected in the NM and lung but not the liver of TG/Cyp2f2-null mice, at levels 10-and 40-fold, respectively, lower than that of mouse CYP2F2 in the TG mice. CYP2B6 protein was detected in the liver (ϳ0.2 pmol/mg of microsomal protein) but not the NM or lung (with a detection limit of 0.04 pmol/mg of microsomal protein) of the TG mice. At least one transgenic protein (CYP2A13) seems to be active, because the NM of the TG mice had greater in vitro and in vivo activities in bioactivation of a CYP2A13 substrate, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (a lung carcinogen), than did the NM of wild-type mice.

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Section: Methodsmentioning

confidence: 99%

Generation and Characterization of aCYP2A13/2B6/2F1-Transgenic Mouse Model

Wei

Lei

et al. 2012

Drug Metab Dispos

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“…BAC modifi cations were verifi ed by DNA sequencing. BAC transgenic constructs were purifi ed independently for microinjection by using a previously described procedure ( 22 ), with slight modifi cation. BAC transgenic constructs were extracted from 250 ml of Escherichia coli ( E. coli ) culture by using a Nucleobond Plasmid Purifi cation kit (Macherey-Nagel, Diiren, Germany).…”

Section: Expression Of Guanylin and Gc-c Mrna In Peritoneal Macrophagesmentioning

confidence: 99%

Involvement of guanylin and GC-C in rat mesenteric macrophages in resistance to a high-fat diet

Akieda-Asai

Sugiyama

Miyazawa

et al. 2013

Journal of Lipid Research

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The worldwide incidence of obesity has accelerated during the last decade. Obesity is caused by excessive accumulation of white adipose tissue, often as the result of ingesting calories in excess of daily requirements ( 1, 2 ). In investigations of disease outcome, excess adipose tissue is defi ned by using the body mass index (BMI), which is calculated as weight (kg)/height (m 2 ). A BMI of 25.0-25.9 kg/m 2 corresponds to an overweight condition, whereas obesity is defi ned as a BMI of 30 kg/m 2 or greater ( 3, 4 ). Adipose depots in mammals are distributed throughout the body and include the fat surrounding the heart and the subcutaneous, retroperitoneal, and mesenteric fat. The amount of mesenteric fat is thought to be correlated most strongly with morbidity rate in obesity ( 5 ). Therefore, investigating the system regulating adipocytes in the mesenteric fat may yield insight into target molecules for treating or preventing obesity-related diseases.Both humans and animals vary in their body-weight responses to high-fat diets (HFDs). When animals are fed HFDs, most of them increase in body weight, with higher levels of adiposity than occur when standard chow is fed. However, a few subjects fed HFDs show less weight gain than do control animals fed standard chow or obesity-prone (diet-induced obesity, DIO) animals. These animals that do not become obese even when fed HFDs are categorized as being "diet resistant" (DR) ( 6, 7 ). To investigate the characteristics of genes expressed in the mesenteric fat tissues, we Abstract A high-fat diet (HFD) is a well-known contributing factor in the development of obesity. Most rats fed HFDs become obese. Those that avoid obesity when fed HFDs are considered diet resistant (DR). We performed a microarray screen to identify genes specifi c to the mesenteric fat of DR rats and revealed high expression of guanylin and guanylyl cyclase C (GC-C) in some subjects. Our histologic studies revealed that the cellular source of guanylin and GC-C is macrophages. Therefore, we developed double-transgenic (Tg) rats overexpressing guanylin and GC-C in macrophages and found that they were resistant to the effects of HFDs. In the mesenteric fat of HFD-fed Tg rats, Fas and perilipin mRNAs were downregulated, and those of genes involved in fatty acid oxidation were upregulated, compared with the levels in HFD-fed wild-type rats. In vitro studies demonstrated that lipid accumulation was markedly inhibited in adipocytes cocultured with macrophages expressing guanylin and GC-C and that this inhibition was reduced after treatment with guanylin-and GC-C-specifi c siRNAs. Our results suggest that the macrophagic guanylin-GC-C system contributes to the altered expression of genes involved in lipid metabolism, leading to resistance to obesity.

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“…1E) was linearized with Not I-HF (NEB, Ipswich, MA), which removes the vector, and isolated after pulsed-field gel electrophoresis and b-agarase (NEB) digestion, according to a published method (Abe et al, 2004). Transgenic mice were produced at the Transgenic and Knockout Core Facility at the Wadsworth Center, as described for the production of the CYP2A13/2B6/2F1-TG model (Wei et al, 2012).…”

Section: Methodsmentioning

confidence: 99%

Generation and Characterization of a Novel CYP2A13-Transgenic Mouse Model

Jia

Liu

et al. 2014

Drug Metab Dispos

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CYP2A13, CYP2B6, and CYP2F1 are neighboring cytochrome P450 genes on human chromosome 19, and the enzymes that they encode overlap in substrate specificity. A CYP2A13/2B6/2F1-transgenic mouse, in which CYP2A13 and 2F1 are both expressed in the respiratory tract and CYP2B6 is expressed in the liver, was recently generated. We generated a CYP2A13 (only) transgenic mouse so that the specific activity of CYP2A13 can be determined. The CYP2B6 and CYP2F1 genes in the CYP2A13/2B6/2F1 genomic clone were inactivated via genetic manipulations, and CYP2A13 was kept intact. A CYP2A13 (only) transgenic (2A13-TG) mouse was generated using the engineered construct and then characterized to confirm transgene integrity and determine copy numbers. The 2A13-TG mice were normal in gross morphology, development, and fertility. As in the CYP2A13/2B6/2F1-transgenic mouse, CYP2A13 expression in the 2A13-TG mouse was limited to the respiratory tract; in contrast, CYP2B6 and 2F1 proteins were not detected. Additional studies using the CYP2A13-humanized (2A13-TG/Cyp2abfgs-null) mouse produced by intercrossing between 2A13-TG and Cyp2abfgs-null mice confirmed that the transgenic CYP2A13 is active in the bioactivation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a lung procarcinogen. The 2A13-TG mouse should be valuable for assessing specific roles of human CYP2A13 in xenobiotic toxicity in the respiratory tract.

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Establishment of an Efficient BAC Transgenesis Protocol and its Application to Functional Characterization of the Mouse Brachyury Locus

Cited by 29 publications

References 23 publications

Generation and Characterization of aCYP2A13/2B6/2F1-Transgenic Mouse Model

Generation and Characterization of aCYP2A13/2B6/2F1-Transgenic Mouse Model

Involvement of guanylin and GC-C in rat mesenteric macrophages in resistance to a high-fat diet

Generation and Characterization of a Novel CYP2A13-Transgenic Mouse Model

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