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Genomic Integration of Lambda EG10 Transgene in Gpt Delta Transgenic Rodents

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Genomic Integration of Lambda EG10 Transgene in Gpt Delta Transgenic Rodents

Kenichi Masumura et al. Genes Environ.

Abstract

Background: Transgenic gpt delta mouse and rat models were developed to perform gpt and Spi(-) assays for in vivo mutagenicity tests. The animals were established by integration of lambda EG10 phage DNA as a transgene into the genome. The inserted position of the transgene on chromosome was determined by fluorescent in situ hybridization and Southern blot analyses; however, the exact position and sequence of the inserted junction were not known. To identify the site and pattern of genomic integration of the transgene copies, genomic DNAs extracted from C57BL/6J gpt delta mice and F344 gpt delta rats were applied to whole genome sequencing and mate-pair analysis.

Results: The result confirmed that multi-copy lambda EG10 transgenes are inserted at a single position in the mouse chromosome 17. The junction contains 70 bp of overlapped genomic sequences, and it has short homology at both ends. A copy number analysis suggested that the inserted transgenes may contain 41 head-to-tail junctions and 16 junctions of other types such as rearranged abnormal junctions. It suggested that the number of intact copies could be approximately 40 at maximum. In the F344 gpt delta rats, transgenes are inserted at a single position in the rat chromosome 4. The junction contains no overlapped sequence but 72-kb genomic sequence including one gene was deleted. The inserted transgenes may contain 15 head-to-tail junctions and two rearranged junctions. It suggested that the number of intact copies could be 14 at maximum. One germline base substitution in the gpt gene rescued from gpt delta rats was characterized.

Conclusions: The exact inserted positions of the lambda EG10 transgene in the genome of gpt delta transgenic rodents were identified. The copy number and arrangement of the transgene were analyzed. PCR primers for quick genotyping of gpt delta mice and rats have been designed.

Keywords: Copy number analysis; Genomic rearrangement; Next generation sequencer; gpt delta mouse and rat.

Figures

Fig. 1
Fig. 1
A brief protocol of identification of the insertion site of lambda EG10 in mouse chromosome
Fig. 2
Fig. 2
Insertion site of lambda EG10 in the mouse genome. Sequenced MPs carrying lambda EG10 and mouse chromosome sequences in each read were selected, and the mouse sequence reads were mapped on the reference mouse genome. The lambda EG10 transgene was integrated into chromosome 17
Fig. 3
Fig. 3
Sequence of the inserted junction of transgenes in gpt delta mouse genome. The white arrow represents the lambda EG10 transgene, and the dark arrow represents the mouse chromosome. Small arrows show the duplicated sequences in the mouse chromosome. *The short chromosome sequences are duplicated at the both junctions. (Chr17_40878810 to 40878879: 70 bps)
Fig. 4
Fig. 4
Number of junctions of lambda EG10 transgenes in gpt delta mice. The number of each junction was estimated by dividing the number of MPs with the average number of upstream and downstream MPs, i.e., 39.5
Fig. 5
Fig. 5
Schematic representation of genomic integration of the EG10 copies in gpt delta mouse. This figure represents a conceptual diagram of integration pattern of the EG10 copies in the genome. Sequential order of each EG10 copy is not identified. Stripe box at both ends represents mouse chromosome sequence. Thick arrows represent the EG10 copies and direction of the sequence. White arrows are intact EG10 copies. Dark arrows are rearranged inactive copies. Line arrows point the junctions between copies or between chromosome and EG10. Small lines represent the MPs covering the junctions. The calculated number of junctions is indicated in a parenthesis
Fig. 6
Fig. 6
Genotyping of gpt delta mouse by PCR. Sequences and positions of PCR primers are presented (a). PCR condition is described in Materials and Methods. Image of agarose gel electrophoresis (b)
Fig. 7
Fig. 7
Insertion site of lambda EG10 in the rat genome. Sequenced MPs carrying lambda EG10 and rat chromosome sequences in each read were selected, and rat sequence reads were mapped on the reference rat genome. The lambda EG10 transgene was integrated into chromosome 4
Fig. 8
Fig. 8
Sequence of the inserted junction of transgenes in the gpt delta rat genome. The white arrow represents the lambda EG10 transgene, and the dark arrow represents the rat chromosome. *The chromosome sequences are deleted at the junction. (Chr4_79828427 to 79900397: 71,789 bps)
Fig. 9
Fig. 9
Number of junctions of lambda EG10 transgenes in gpt delta rats. The number of each junction was estimated by dividing the number of MPs with the average number of upstream and downstream MPs, i.e., 77
Fig. 10
Fig. 10
Schematic representation of genomic integration of the EG10 copies in gpt delta rat. This figure represents a conceptual diagram of integration pattern of the EG10 copies in the genome. Sequential order of each EG10 copy is not identified. Stripe box at both ends represents rat chromosome sequence. Thick arrows represent the EG10 copies and direction of the sequence. White arrows are intact EG10 copies. Dark arrows are rearranged inactive copies. Line arrows point the junctions between copies or between chromosome and EG10. Small lines represent the mate pairs (MPs) covering the junctions. The calculated number of junctions is indicated in a parenthesis
Fig. 11
Fig. 11
Genotyping of gpt delta rats by PCR. Sequences and positions of PCR primers are presented (a). PCR condition is described in Materials and Methods. Image of agarose gel electrophoresis (b)
Fig. 12
Fig. 12
Characterization of the phenotype of the gpt mutants rescued from gpt delta rats by spot testing. The cultured gpt mutant clones were spotted onto M9 with Cm plates (a) and M9 with Cm and 6TG plates (b) in dilution series. The experimental condition was described in Materials and Methods. Lane 1 and 2: Clones which have the gpt mutation at 299 T to A. Lane 3 and 4: Clones which have no gpt mutation. Lane 5 and 6: Positive control which has a gpt mutation (185 G to A in lane 5 and 416–418 GGG to GG in lane 6). Lane 7: Negative control which has no gpt mutation. The A:T to T:A transversion at position 299 did not alter 6TG sensitivity as a negative control. Positive control gpt mutants exhibited 6TG resistance

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