Genomic integration of lambda EG10 transgene in gpt delta transgenic rodents

Abstract

BackgroundTransgenic 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.ResultsThe 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s41021-015-0024-6) contains supplementary material, which is available to authorized users.

Mutagenicity of Helicobacter hepaticus infection in the lower bowel mucosa of 129/SvEv Rag2−/‐Il10−/−gpt delta mice is influenced by sex

“…A λ‐EG10‐based transgenic C57BL/6 mouse model was specifically designed to facilitate in vivo detection of genomic point mutations and large deletions . It has been estimated by whole genome sequencing that C57BL/6 gpt delta mice carry tandem arrays of approximately 40 copies of the bacterial guanine phosphoribosyltransferase gene ( gpt ) at a single site on chromosome 17 . In this experimental in vivo system, point mutations can be detected and quantified by 6‐thioguanine (6‐TG) selection, whereas deletions up to 10 kb in length are characterized by selection based on sensitivity to P2 interference (Spi − ) .…”

“…15 It has been estimated by whole genome sequencing that C57BL/6 gpt delta mice carry tandem arrays of approximately 40 copies of the bacterial guanine phosphoribosyltransferase gene ( gpt) at a single site on chromosome 17. 16 In this experimental in vivo system, point mutations can be detected and quantified by 6-thioguanine (6-TG) selection, whereas deletions up to 10 kb in length are characterized by selection based on sensitivity to P2 interference (Spi − ). 15 We previously demonstrated that H. pylori SS1 infection in C57BL/6 gpt delta mice promoted point mutation frequencies in gastric mucosa in females but not in males compared to uninfected controls, whereas no difference in large deletions was noted.…”

Estimation of the frequency of inherited germline mutations by whole exome sequencing in ethyl nitrosourea-treated and untreated gpt delta mice

“…If ENU-induced germline gpt mutations are transmitted to offspring, then a very high gpt mutant frequency could be observed in the F1 mice. It has been reported that there are approximately 40 copies of each transgene per haploid in gpt delta mice [ 38 ]. If one copy contains the inherited gpt mutation, then the expected mutant frequency in the F1 mice would be 1/80 = 12.5 × 10 −3 , which is 1000 times higher than the control level.…”

Tissue‐specific and time‐dependent clonal expansion of ENU‐induced mutant cells in gpt delta mice

“…The mutation frequency was calculated by dividing the number of unique mutations by the total number of colonies (for all calculations, the respective dilution factors were taken into account). Furthermore, to focus on the kinetics of mouse cell survival after exposure to ENU, the mutant frequency per cell was calculated based on 40 copies of the transgene per cell [Masumura et al, ]. Each cell was assumed to contain no more than one mutant transgene in its 40 transgene cassette; thus we converted mutant transgene frequency to mutant cell frequency as follows:…”