| The establishment of transgenic animal models for mutation detection makes it possible to study the induced DNA damage, DNA repair,mutagenesis and carcinogenesis in vivo and provides a rapid and effective four-dimension detection system for the tissue-specific mutation. At present, transgenic mouse, marked as BigBlueTM,MutaTMMouse and Xenomouse, have come into commercial market.The BigBlueTM or MutaTMMouse, based on the package of λvector, has the limitation of the weight of packged DNA molecular When rescuing the λvector DNA. Although Xenomouse takes over this shortage, the longer LacI gene is used as target gene, it is very time-consuming when sequencing and easy to go wrong when screening the mutants based on the colony color.Our group has been involving in the study of transgenic animal mutation detection system for a long time. In this paper, a novel transgenic animal mutation detection system, pMCLacI/neo plasmid-based transgenic mouse was established. pMCLacI/neo plasmid contains 2 copies of LacI gene: One is in expression state, the other in silence state, thereby would imitate the states of expressed and non expressed genes in vivo respectively. So when introduced in vivo, pMCLacI/neo can detect the mutation of both expression and silence gene simultaneously. This system could be applied for genetic toxicity control of chemicals in vivo, study of mechanism of carcinogenesis and progression, evaluation of drug security and so on. Moreover, this system would provide better rationale for risk assessment of potential mutagens and carcinogens.The pMCLacI/neo transgenic mouse contains two LacI target genes in different states, different from the other systems , which have target gene in only one state. So seeking for a rapid ,effective way to detect this two states of target genes, is very important. In this research ,a positive selection system M9/L is used to separate the two LacI target genes in pMCLacI/neo. Some LacI mutants are used as positive control to test the feasibility of this M9/L positive system. The results suggested thatit is able to detect both the two LacI target genes in pMCLacI/neo ,demonstrating that M9/L positive system is effective and efficient in detecting the target mutations.Based on the established pMCLacI/neo transgenic mouse, we detected the tissue-specific mutation frequencies and spectrum induced by MNNG. In 25 identified mutants, 13 mutants exist at the -COOH of LacI to LacO region and 7 are located at the -NH2 of LacI coding region , both are hot spots. Analysis of the expression state of mutant genes showed that 24%(6/25) is expression target genes and 76% mutation is silence target genes. Most mutations is in the CpG island, which indicate again that the CpG island is the dominant mutation region in the eukaryotic genomic DNA. As the pMCLacI/neo shuttle vector is a novel bi-functional mutation detection system, apart from be used in above transgenic animal study , it was also used in our study of MNNG-indued DNA damage and repair research. In this paper, MGMT(O6-methylguanine-DNA methyltransferase)was disrupted or over-expressed as well in NIH3T3 cells. The corresponding mutation spectrums induced by MNNG were detected. Alkylation of DNA at the O6-position of guanine is one of the most critical events leading to induction of mutation as well as to cancer.The enzyme O6-methylguanine-DNA methyltransferase repairs this and related lesions in DNA.By means of gene targeting and shuttle vector,we established mouse cell lines deficient in the methyltransferase activity Administration of methylnitrosourea (MNNG) to these gene-targeted NIH3T3 cells, the cells showed high sensitivity.Flow cytometric analyses revealed that ,the cells undergo apoptotic death ,which occurs after G2-M arrest in the second cycle of cell proliferation.The establishment of MGMT knock-out mutation detection system in NIH3T3 ,would help us in the related study of MGMT. Meanwhile , some alkylating agents as chemo therapy drug are applied to the treatment of some...
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