title: Cre/loxP-regulated transgenic zebrafish model for neural progenitor-specific oncogenic Kras expression.
abstract: Ras proteins regulate signaling pathways that control many cellular responses, such as proliferation, survival, and differentiation. However, there are intriguing questions about the relationship between the developmental timing of specific mutations and the resultant phenotypes in individual cells. In this study, we used the Cre/loxP system for maintaining transgenic zebrafish lines harboring oncogenic Kras(V12) under the nestin promoter, and investigated the developmental effects of Ras activation in neural progenitor cells. Activated human Kras(V12) was induced within pDSNesLCherryLEGFPKRas(V12) transgenic fish by Cre mRNA injection. Cre-mediated gene excision was confirmed by polymerase chain reaction, and the injected embryos were investigated for Kras(V12) effects using the hemotoxylin-eosin staining, terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling assay, and in situ hybridization. pDSNesLCherryLEGFPKRas(V12) transgenic embryos normally expressed mCherry in their central nervous system throughout the developmental stage. However, Cre mRNA injection efficiently excised the flanking stop sequence, and the injected embryos expressed enhanced green fluorescent protein in their brain with severe edema. Brain histology showed that neuronal cell differentiation could occur in spite of oncogenic Kras(V12) overexpression, but massive apoptosis and brain edema caused early embryonal death. In summary, the overexpression of Kras(V12) induces extensive apoptosis of neural progenitor cells followed by severe edema of the brain. However, some neural progenitor cells are resistant to Kras(V12) and can retain their ability to differentiate into neurons. Finally, our transgenic model demonstrates the inability of Kras(V12) alone to induce brain tumors at the early stage of development.
authors: Institute for Experimental Animals, College of Medicine, Seoul National University, Seoul, Korea.
authors: Seung-Hyeok
authors: SH
authors: Seok
authors: Yi-Rang
authors: YR
authors: Na
authors: Ju-Hee
authors: JH
authors: Han
authors: Tae-Hyoun
authors: TH
authors: Kim
authors: Hyun
authors: H
authors: Jung
authors: Byoung-Hee
authors: BH
authors: Lee
authors: Alexander
authors: A
authors: Emelyanov
authors: Serguei
authors: S
authors: Parinov
authors: Jae-Hak
authors: JH
authors: Park
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D011905:Genes, ras; D006801:Humans; D019426:Integrases / Q000235:genetics / Q000502:physiology*; D007381:Intermediate Filament Proteins / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000235:genetics; D064231:Nestin; D009474:Neurons / Q000166:cytology* / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D011995:Recombination, Genetic; D013234:Stem Cells / Q000378:metabolism*; D015027:Zebrafish; D018631:ras Proteins / Q000235:genetics*
chemical_list: D007381:Intermediate Filament Proteins; C117307:KRAS protein, human; C577484:NES protein, human; D009419:Nerve Tissue Proteins; D064231:Nestin; D011518:Proto-Oncogene Proteins; C045073:Cre recombinase; D019426:Integrases; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins