Two base pair deletion in IL2 receptor γ gene in NOD/SCID mice induces a highly severe immunodeficiency / 한국실험동물학회지

Genome editing has recently emerged as a powerful tool for generating mutant mice. Small deletions of nucleotides in the target genes are frequently found in CRISPR/Cas9 mediated mutant mice. However, there are very few reports analyzing the phenotypes in small deleted mutant mice generated by CRISPR/Cas9. In this study, we generated a mutant by microinjecting sgRNAs targeting the IL2 receptor γ gene and Cas9 protein, into the cytoplasm of IVF-derived NOD.CB17/Prkdcscid/JKrb (NOD/SCID) mice embryos, and further investigated whether a 2 bp deletion of the IL2 receptor γ gene affects severe deficiency of immune cells as seen in NOD/LtSz-scid IL2 receptor γ−/− (NSG) mice. Our results show that the thymus weight of mutant mice is significantly less than that of NOD/SCID mice, whereas the spleen weight was marginally less. T and B cells in the mutant mice were severely deficient, and NK cells were almost absent. In addition, tumor growth was exceedingly increased in the mutant mice transplanted with HepG2, Raji and A549 cells, but not in nude and NOD/SCID mice. These results suggest that the NOD/SCID mice with deletion of 2 bp in the IL2 receptor γ gene shows same phenotype as NSG mice. Taken together, our data indicates that small deletions by genome editing is sufficient to generate null mutant mice.

Comparison of commonly used ICR stocks and the characterization of Korl:ICR / 한국실험동물학회지

Mouse is a commonly used animal in life science studies and is classified as outbred if genetically diverse and inbred if genetically homogeneous. Outbred mouse stocks, are used in toxicology, oncology, infection and pharmacology research. The National Institute of Food and Drug Safety Evaluation (NIFDS; former the Korea National Institute of Health) have bred ICR mice for more than 50 years. We investigated to provide users with information and promote accountability to the Korl:ICR. To secure the indigenous data, biological characteristics of Korl:ICR were identified by comparing with other ICR stocks. This domestic ICR stock was denominated as ‘Korl:ICR’. Phylogenetic analysis using SNPs indicated that the population stratification of the Korl:ICR was allocated different area with other ICR. In addition, we measured litter size, body weight, body length, various organ weight, hematology and clinical blood chemistry of the Korl:ICR compared to other ICR. Otherwise, there are no significant differences among the biological phenotypes of Korl:ICR and other ICR. These results suggest that as a genetically indigenous source colony, the Korl:ICR is seperated (or independent) stock with other ICR. Also, we confirmed that there is no difference among the Korl:ICR and other ICR on biological phenotypes. Therefore, the Korl:ICR source colony might be a new stock in distinction from other ICR, it is a good milestone in securing ownership of the national laboratory animal resource. The NIFDS expects that the Korl:ICR mice will be useful animal resource for our domestic researchers.

An important role for peroxiredoxin II in survival of A549 lung cancer cells resistant to gefitinib

Redox adaptation is an important concept that explains the mechanisms by which cancer cells survive under persistent endogenous oxidative stress and become resistant to certain anticancer agents. To investigate this concept, we determined the expression levels of peroxiredoxins (Prxs), antioxidant enzymes in drug-resistant non-small cell lung carcinoma cells. Prx II was remarkably increased only in A549/GR (gefitinib-resistant) cells compared with A549 cells, consistent with methylation/demethylation. Prx II was highly methylated in the A549 cells but was demethylated in the A549/GR cells. The elevated expression of Prx II resulted in the downregulation of reactive oxygen species (ROS) and cell death and upregulation of cell cycle progression in the A549/GR cells. When Prx II mRNA in the A549/GR cells was knocked down, the levels of ROS and apoptosis were significantly recovered to the levels of the controls. In addition, signaling molecules involved in apoptosis were increased in the A549/GR-shPrx II cells. There was no difference in the expression of MAPK/ERK between the A549/GR cells and A549/GR-shPrx II cells, but the phosphorylation of JNK was increased in the A549/GR cells and was markedly decreased in the A549/GR-shPrx II cells. Colony number and tumor growth were significantly decreased in the A549/GR-shPrx II cells compared with the A549/GR cells. Our findings suggest that Prx II has an important role in cancer cell survival via the modulation of signaling molecules involved in apoptosis and the phosphorylation of JNK by the downregulation of ROS levels in A549/GR cells.

An important role for peroxiredoxin II in survival of A549 lung cancer cells resistant to gefitinib

Redox adaptation is an important concept that explains the mechanisms by which cancer cells survive under persistent endogenous oxidative stress and become resistant to certain anticancer agents. To investigate this concept, we determined the expression levels of peroxiredoxins (Prxs), antioxidant enzymes in drug-resistant non-small cell lung carcinoma cells. Prx II was remarkably increased only in A549/GR (gefitinib-resistant) cells compared with A549 cells, consistent with methylation/demethylation. Prx II was highly methylated in the A549 cells but was demethylated in the A549/GR cells. The elevated expression of Prx II resulted in the downregulation of reactive oxygen species (ROS) and cell death and upregulation of cell cycle progression in the A549/GR cells. When Prx II mRNA in the A549/GR cells was knocked down, the levels of ROS and apoptosis were significantly recovered to the levels of the controls. In addition, signaling molecules involved in apoptosis were increased in the A549/GR-shPrx II cells. There was no difference in the expression of MAPK/ERK between the A549/GR cells and A549/GR-shPrx II cells, but the phosphorylation of JNK was increased in the A549/GR cells and was markedly decreased in the A549/GR-shPrx II cells. Colony number and tumor growth were significantly decreased in the A549/GR-shPrx II cells compared with the A549/GR cells. Our findings suggest that Prx II has an important role in cancer cell survival via the modulation of signaling molecules involved in apoptosis and the phosphorylation of JNK by the downregulation of ROS levels in A549/GR cells.