Mouse genetics: Catalogue and scissors

Phenotypic analysis of gene-specific knockout (KO) mice has revolutionized our understanding of in vivo gene functions. As the use of mouse embryonic stem (ES) cells is inevitable for conventional gene targeting, the generation of knockout mice remains a very time-consuming and expensive process. To accelerate the large-scale production and phenotype analyses of KO mice, international efforts have organized global consortia such as the International Knockout Mouse Consortium (IKMC) and International Mouse Phenotype Consortium (IMPC), and they are persistently expanding the KO mouse catalogue that is publicly available for the researches studying specific genes of interests in vivo. However, new technologies, adopting zinc-finger nucleases (ZFNs) or Transcription Activator-like Effector (TALE) Nucleases (TALENs) to edit the mouse genome, are now emerging as valuable and effective shortcuts alternative for the conventional gene targeting using ES cells. Here, we introduce the recent achievement of IKMC, and evaluate the significance of ZFN/TALEN technology in mouse genetics. [BMB Reports 2012; 45(12): 686-692]

P-glycoprotein confers acquired resistance to 17-DMAG in lung cancers with an ALK rearrangement

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.Background Because anaplastic lymphoma kinase (ALK) is dependent on Hsp90 for protein stability, Hsp90 inhibitors are effective in controlling growth of lung cancer cells with ALK rearrangement. We investigated the mechanism of acquired resistance to 17-(Dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG), a geldanamycin analogue Hsp90 inhibitor, in H3122 and H2228 non-small cell lung cancer cell lines with ALK rearrangement. Methods Resistant cell lines (H3122/DR-1, H3122/DR-2 and H2228/DR) were established by repeated exposure to increasing concentrations of 17-DMAG. Mechanisms for resistance by either NAD(P)H/quinone oxidoreductase 1 (NQO1), previously known as a factor related to 17-DMAG resistance, or P-glycoprotein (P-gp; ABCB1/MDR1) were queried using RT-PCR, western blot analysis, chemical inhibitors, the MTT cell proliferation/survival assay, and cellular efflux of rhodamine 123. Results The resistant cells showed no cross-resistance to AUY922 or ALK inhibitors, suggesting that ALK dependency persists in cells with acquired resistance to 17-DMAG. Although expression of NQO1 was decreased in H3122/DR-1 and H3122/DR-2, NQO1 inhibition by dicumarol did not affect the response of parental cells (H2228 and H3122) to 17-DMAG. Interestingly, all resistant cells showed the induction of P-gp at the protein and RNA levels, which was associated with an increased efflux of the P-gp substrate rhodamine 123 (Rho123). Transfection with siRNA directed against P-gp or treatment with verapamil, an inhibitor of P-gp, restored the sensitivity to the drug in all cells with acquired resistance to 17-DMAG. Furthermore, we also observed that the growth-inhibitory effect of 17-DMAG was decreased in A549/PR and H460/PR cells generated to over-express P-gp by long-term exposure to paclitaxel, and these cells recovered their sensitivity to 17-DMAG through the inhibition of P-gp. Conclusion P-gp over-expression is a possible mechanism of acquired resistance to 17-DMAG in cells with ALK rearrangement

Immunohistochemical identification and quantitative analysis of cytoplasmic Cu/Zn superoxide dismutase in mouse organogenesis

Cytoplasmic Cu/Zn superoxide dismutase (SOD1) is an antioxidant enzyme that converts superoxide to hydrogen peroxide in cells. Its spatial distribution matches that of superoxide production, allowing it to protect cells from oxidative stress. SOD1 deficiencies result in embryonic lethality and a wide range of pathologies in mice, but little is known about normal SOD1 protein expression in developing embryos. In this study, the expression pattern of SOD1 was investigated in post-implantation mouse embryos and extraembryonic tissues, including placenta, using Western blotting and immunohistochemical analyses. SOD1 was detected in embryos and extraembryonic tissues from embryonic day (ED) 8.5 to 18.5. The signal in embryos was observed at the lowest level on ED 9.5-11.5, and the highest level on ED 17.5-18.5, while levels remained constant in the surrounding extraembryonic tissues during all developmental stages examined. Immunohistochemical analysis of SOD1 expression on ED 13.5-18.5 revealed its ubiquitous distribution throughout developing organs. In particular, high levels of SOD1 expression were observed in the ependymal epithelium of the choroid plexus, ganglia, sensory cells of the olfactory and vestibulocochlear epithelia, blood cells and vessels, hepatocytes and hematopoietic cells of the liver, lymph nodes, osteogenic tissues, and skin. Thus, SOD1 is highly expressed at late stages of embryonic development in a cell- and tissue-specific manner, and can function as an important antioxidant enzyme during organogenesis in mouse embryos

Effects of endocrine disrupting chemicals on expression of phospholipid hydroperoxide glutathione peroxidase mRNA in rat testes

Phospholipid hydroperoxide glutathione peroxidase (PHGPx), an antioxidative selenoprotein, is modulated by estrogen in the testis and oviduct. To examine whether potential endocrine disrupting chemicals (EDCs) affect the microenvironment of the testes, the expression patterns of PHGPx mRNA and histological changes were analyzed in 5-week-old Sprague-Dawley male rats exposed to several EDCs such as an androgenic compound [testosterone (50, 200, and 1,000 µg/kg)], anti-androgenic compounds [flutamide (1, 5, and 25 mg/kg), ketoconazole (0.2 and 1 mg/kg), and diethylhexyl phthalate (10, 50, and 250 mg/kg)], and estrogenic compounds [nonylphenol (10, 50, 100, and 250 mg/kg), octylphenol (10, 50, and 250 mg/kg), and diethylstilbestrol (10, 20, and 40 µg/kg)] daily for 3 weeks via oral administration. Mild proliferation of germ cells and hyperplasia of interstitial cells were observed in the testes of the flutamide-treated group and deletion of the germinal epithelium and sloughing of germ cells were observed in testes of the diethylstilbestrol-treated group. Treatment with testosterone was shown to slightly decrease PHGPx mRNA levels in testes by the reverse transcriptionpolymerase chain reaction. However, anti-androgenic compounds (flutamide, ketoconazole, and diethylhexyl phthalate) and estrogenic compounds (nonylphenol, octylphenol, and diethylstilbestrol) significantly upregulated PHGPx mRNA in the testes (p < 0.05). These findings indicate that the EDCs might have a detrimental effect on spermatogenesis via abnormal enhancement of PHGPx expression in testes and that PHGPx is useful as a biomarker for toxicity screening of estrogenic or antiandrogenic EDCs in testes

PIERCE1 is critical for specification of left-right asymmetry in mice

The specification of left-right asymmetry of the visceral organs is precisely regulated. The earliest breakage of left-right symmetry occurs as the result of leftward flow generated by asymmetric beating of nodal cilia, which eventually induces asymmetric Nodal/Lefty/Pitx2 expression on the left side of the lateral plate mesoderm. PIERCE1 has been identified as a p53 target gene involved in the DNA damage response. In this study, we found that Pierce1-null mice exhibit severe laterality defects, including situs inversus totalis and heterotaxy with randomized situs and left and right isomerisms. The spectrum of laterality defects was closely correlated with randomized expression of Nodal and its downstream genes, Lefty1/2 and Pitx2. The phenotype of Pierce1-null mice most closely resembled that of mutant mice with impaired ciliogenesis and/or ciliary motility of the node. We also found the loss of asymmetric expression of Cerl2, the earliest flow-responding gene in the node of Pierce1-null embryos. The results suggest that Pierce1-null embryos have defects in generating a symmetry breaking signal including leftward nodal flow. This is the first report implicating a role for PIERCE1 in the symmetry-breaking step of left-right asymmetry specification

Loss of progesterone receptor membrane component 1 promotes hepatic steatosis via the induced de novo lipogenesis

Abstract Non-alcoholic fatty liver disease (NAFLD) results from triglyceride accumulation within the liver and some of them advances to non-alcoholic steatohepatitis (NASH). It is important to note that in NAFLD development, hepatic de novo lipogenesis (DNL) derives from excess carbohydrates and fats under a condition of excess energy through β-oxidation. As a main regulator for DNL, sterol regulatory element-binding protein 1 (Srebp-1) forms complex with progesterone receptor membrane component 1 (Pgrmc1). To investigate whether Pgrmc1 may have a notable effect on DNL via SREBP-1 activation, we generated Pgrmc1 knockout (KO) mice and fed a high fat diet for one month. High-fat-fed Pgrmc1 KO mice showed a substantial increase in levels of hepatic TG accumulation, and they were predisposed to NAFLD when compared to WT mice. Loss of Pgrmc1 increased mature SREBP-1 protein level, suggesting that induction of hepatic steatosis in Pgrmc1 KO mice might be triggered by de novo lipogenesis. Moreover, Pgrmc1 KO mice were also more vulnerable to early stage of NASH, showing high levels of alanine aminotransferase, obesity-linked pro-inflammatory cytokines, and fibrosis markers. This is interesting because Pgrmc1 involves with the first step in regulating the hepatic de novo lipogenesis under an excess energy condition

Enhanced Protective Effects of Combined Treatment with β-Carotene and Curcumin against Hyperthermic Spermatogenic Disorders in Mice

Scrotal hyperthermia leads to oxidative stress and apoptosis in spermatogenic cells, which subsequently causes male infertility. In this study, we examined the effects of β-carotene and/or curcumin on heat-stress- (HS-) induced testicular injuries in mice. ICR male mice (8 weeks old) were consecutively treated with β-carotene (10 mg/kg) and/or curcumin (20 mg/kg) orally once a day for 14 days and then subjected to single exposure with scrotal HS at 43°C for 15 min on day 7. HS induced a significant reduction in testicular weight, appearance of multinucleated giant cells, and desquamation of germ cells in destructive seminiferous tubules, as well as degenerative Leydig cells. Moreover, HS reduced the superoxide dismutase (SOD) activity and mRNA levels of mitochondrial SOD, phospholipid hydroperoxide glutathione peroxidase, B-cell lymphoma-extra-large, and 3β-hydroxysteroid dehydrogenase, with increases in lipid peroxidation levels and mRNA levels of BCL2-associated X protein and caspase-3 relative to those of the control group. However, these changes were significantly recovered by combined treatment with β-carotene and curcumin after HS. These findings indicate that the combined treatment with β-carotene and curcumin might be a valuable protective agent to ameliorate hyperthermic spermatogenic disorders via its potent antioxidative, antiapoptotic, and androgen synthetic effects