1992 Fiscal Year Final Research Report Summary
The basic studies on the insertional mutation in transgenic mice
| Project/Area Number |
02680040
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Laboratory animal science
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| Research Institution | Keio University, School of Medicine |
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Principal Investigator |
SHIMODA Kouji Keio University, School of Medicine, Instructor, 医学部, 助手 (00129470)
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| Co-Investigator(Kenkyū-buntansha) |
MAEJIMA Kazuyoshi Keio University, School of Medicine, Professor, 医学部, 教授 (70051464)
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| Project Period (FY) |
1990 – 1992
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| Keywords | transgenic mice / homologous recombination / plasmid rescue |
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| Research Abstract |
To determine how DNA fragments integrate into the host genome after microinjection into the pronuclei of fertilized mouse eggs, we analyzed the recombination status in transgenic mice produced by coinjection with two or three fragments which shared overlap-ping regions each other. Circular plasmid DNA, pSy14 (10.6 kb), which is composed of human papillomavirus type 16 DNA in BamHI site of pUC19 plasmid, was digested with 3 pairs of restriction enzymes and each fragment which possessed overlapping regions in its 5' and 3' ends was purified by agarose gel electrophoresis. Three fragments were mixed together with equal molar amounts and coinjected into fertilized mouse eggs to generate transgenic mice. Southern analysis and plasmid rescue experiments of tail DNA showed that the coinjected fragments were reconstructed to original pSy14 plasmid sequences by homologous recombination in their overlapping regions and then the tandem repeats of result-ant pSy14 integrated into host chromosome. Coinjection with two fragments also produced homologous recombination events in trans-genic mice. Plasmid rescue experiments into Escherichia coli indicated that mutations in the Methylation Dependent Restriction System were required for the efficient recovery of plasmid DNA from transgenic genome into competent cells because genomic sequences were usually methylated in many C residues of the CG motif. Efficiency of plasmid rescue was varied among the tissues from which genomic DNA were isolated.
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