Objective: To establish transgenic mouse lineages containing copies of a stably integratedpSPORT1 plasmid by microinjection into fertilized eggs and to provide an efficient animal model for studyinggene mutations in v...Objective: To establish transgenic mouse lineages containing copies of a stably integratedpSPORT1 plasmid by microinjection into fertilized eggs and to provide an efficient animal model for studyinggene mutations in vivo. Methods and Results: 2594 fertilized eggs from KM white mice were injected withpSPORT1 DNA and transferred into the oviducts of 103 pseudopregnant females. from which 237 offspringswere obtained. 40 of these offsprings were identified positive for the forgeign gene by PCR analysis and 38were reproved positive by Southern blot analysis. Finally, eight of the stout mice whose genornes were integrated with intact pSPORT1 vectors, verified by Southern blotting analysis, were chosen as founders to establish the transgenic mouse lineages. The experimental results inchoated that the pregnant rate of recipientfemales and the Positive rate of offsprings were dramatically influenced by the structure of the transgene(linearized or circular ) and the mode of egg-transfer. The integration rate of linearized transgene was significantly higher than that of the circular transgene,and female with two-side oviduct transfer of fertilized eggs waseasy to have baby mice. Conclusion: (1 ) Transgenic mouse lineages containing copies of a stably integratedPSPORT 1 plasmid were established; (2 ) The linearized transgene and two-side oviduct transfer of fertilizedeggs is more efficient in preparing transgenic mice.展开更多
EMS\|induced mutant frequency and mutation spectrum as well as background mutant frequency have been characterized for bone marrow of the D6\|2 transgenic mice. The \%lacI\% genes carried on pSPORT1 vectors were recov...EMS\|induced mutant frequency and mutation spectrum as well as background mutant frequency have been characterized for bone marrow of the D6\|2 transgenic mice. The \%lacI\% genes carried on pSPORT1 vectors were recovered from the treated or untreated mouse genomic DNA by excision and circularization, and analyzed \%in vitro\% for mutations that occurred in the mouse bone marrow. \%lacI\+-\% mutants were positively selected with the M9/L media. The 6 \%lacI\+-\% mutants were identified out of 11 935 vectors recovered from genomic DNA of the treated mice (mutant frequency was 50×10 -5 ), while no mutant was found in 11 649 vectors from untreated mice (the background mutant frequency was lower than 8.6×10 -5 ). Two regions of \%lacI\% for each mutant, in which the majority of sensitive sites for inactivation of the \%lacI\% gene product have been located, were sequenced and 16 mutation events were identified. The predominant mutations (14/16 or 87.5%) were base substitutions, whereas the remaining 2 mutations were single base deletions (12.5%). Of these base substitutions, transversions made up 9/14 or 64%, and transitions comprised 5/14 or 36%. These findings were markedly different from the spontaneous spectra characterized by using BigBlue system, as well as from the EMS\| induced mutation spectra obtained with \%in vitro\% assay systems, where the EMS\|induced predominant mutations are GC→AT transitions. In addition, 45% of mutations analyzed occurred at CpG dinucleotides, which was in accordance with previous studies with other systems. These data show that: (i) the D6\|2 transgenic mouse lineage is a suitable model for studying mutagenesis \%in vivo\%; (ii) a fundamental difference in mutagenesis for EMS between \%in vitro\% and \%in vivo\% assay systems may exist, but more extensive sequence analyses are required to determine the possible differences in mutation spectra.展开更多
文摘Objective: To establish transgenic mouse lineages containing copies of a stably integratedpSPORT1 plasmid by microinjection into fertilized eggs and to provide an efficient animal model for studyinggene mutations in vivo. Methods and Results: 2594 fertilized eggs from KM white mice were injected withpSPORT1 DNA and transferred into the oviducts of 103 pseudopregnant females. from which 237 offspringswere obtained. 40 of these offsprings were identified positive for the forgeign gene by PCR analysis and 38were reproved positive by Southern blot analysis. Finally, eight of the stout mice whose genornes were integrated with intact pSPORT1 vectors, verified by Southern blotting analysis, were chosen as founders to establish the transgenic mouse lineages. The experimental results inchoated that the pregnant rate of recipientfemales and the Positive rate of offsprings were dramatically influenced by the structure of the transgene(linearized or circular ) and the mode of egg-transfer. The integration rate of linearized transgene was significantly higher than that of the circular transgene,and female with two-side oviduct transfer of fertilized eggs waseasy to have baby mice. Conclusion: (1 ) Transgenic mouse lineages containing copies of a stably integratedPSPORT 1 plasmid were established; (2 ) The linearized transgene and two-side oviduct transfer of fertilizedeggs is more efficient in preparing transgenic mice.
文摘EMS\|induced mutant frequency and mutation spectrum as well as background mutant frequency have been characterized for bone marrow of the D6\|2 transgenic mice. The \%lacI\% genes carried on pSPORT1 vectors were recovered from the treated or untreated mouse genomic DNA by excision and circularization, and analyzed \%in vitro\% for mutations that occurred in the mouse bone marrow. \%lacI\+-\% mutants were positively selected with the M9/L media. The 6 \%lacI\+-\% mutants were identified out of 11 935 vectors recovered from genomic DNA of the treated mice (mutant frequency was 50×10 -5 ), while no mutant was found in 11 649 vectors from untreated mice (the background mutant frequency was lower than 8.6×10 -5 ). Two regions of \%lacI\% for each mutant, in which the majority of sensitive sites for inactivation of the \%lacI\% gene product have been located, were sequenced and 16 mutation events were identified. The predominant mutations (14/16 or 87.5%) were base substitutions, whereas the remaining 2 mutations were single base deletions (12.5%). Of these base substitutions, transversions made up 9/14 or 64%, and transitions comprised 5/14 or 36%. These findings were markedly different from the spontaneous spectra characterized by using BigBlue system, as well as from the EMS\| induced mutation spectra obtained with \%in vitro\% assay systems, where the EMS\|induced predominant mutations are GC→AT transitions. In addition, 45% of mutations analyzed occurred at CpG dinucleotides, which was in accordance with previous studies with other systems. These data show that: (i) the D6\|2 transgenic mouse lineage is a suitable model for studying mutagenesis \%in vivo\%; (ii) a fundamental difference in mutagenesis for EMS between \%in vitro\% and \%in vivo\% assay systems may exist, but more extensive sequence analyses are required to determine the possible differences in mutation spectra.
