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. 2017 May 5;45(8):e62.
doi: 10.1093/nar/gkw1329.

Efficient mapping of transgene integration sites and local structural changes in Cre transgenic mice using targeted locus amplification

Carol Cain-Hom  1 , Erik Splinter  2 , Max van Min  2 , Marieke Simonis  2 , Monique van de Heijning  2 , Maria Martinez  1 , Vida Asghari  1 , J Colin Cox  1 , Søren Warming  3
Affiliations

Efficient mapping of transgene integration sites and local structural changes in Cre transgenic mice using targeted locus amplification

Carol Cain-Hom et al. Nucleic Acids Res. .

Abstract

Cre/LoxP technology is widely used in the field of mouse genetics for spatial and/or temporal regulation of gene function. For Cre lines generated via pronuclear microinjection of a Cre transgene construct, the integration site is random and in most cases not known. Integration of a transgene can disrupt an endogenous gene, potentially interfering with interpretation of the phenotype. In addition, knowledge of where the transgene is integrated is important for planning of crosses between animals carrying a conditional allele and a given Cre allele in case the alleles are on the same chromosome. We have used targeted locus amplification (TLA) to efficiently map the transgene location in seven previously published Cre and CreERT2 transgenic lines. In all lines, transgene insertion was associated with structural changes of variable complexity, illustrating the importance of testing for rearrangements around the integration site. In all seven lines the exact integration site and breakpoint sequences were identified. Our methods, data and genotyping assays can be used as a resource for the mouse community and our results illustrate the power of the TLA method to not only efficiently map the integration site of any transgene, but also provide additional information regarding the transgene integration events.

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Figures

Figure 1.
Figure 1.
Copy number variation (CNV) analysis for all seven Cre/CreERT2 lines described in this study. Y-axis show calculated copy number by comparison to a homozygous (2 copies) Cre knock-in mouse control. Animals carrying the Cre transgene were first identified and subsequently CNV was performed to determine allelic status. Each data point represents one animal. Cq values used to calculate ΔCq, ΔΔCq and copy number were mean of values from 3–4 technical replicates. TG/TG: homozygous transgenic. TG/WT: hemizygous transgenic.
Figure 2.
Figure 2.
Flow diagram of the TLA process. For details, see the ‘Materials and Methods’ section.
Figure 3.
Figure 3.
TLA coverage and analysis plots. Upper panels, TLA sequence coverage: mouse chromosomes 1 through X are arranged on the Y-axis. X-axis shows chromosomal position. A detailed view of TLA sequence coverage surrounding the integration site is expanded in the middle panels. Lower panels: graphic representation of transgene integration site and structural changes. Gray: flanking genomic sequence. Blue: transgene and corresponding genomic coordinates of the transgene sequence (mouse, human or rat genome). Only one transgene copy is shown for simplicity. Green: DNA sequences that cannot be mapped to a genome (e.g. vector sequence). Red: a 40 kb mouse Vat1l sequence co-integrated with the Cdh5-CreERT2 transgene and a 142 kb mouse Chr1 sequence co-integrated with the Syn1-Cre transgene. Duplicated regions are highlighted by hatching. For Syn1-Cre, one end of the duplicated region is unknown (indicated by a dotted line). ND: no data. Mouse genome assembly: mm9; human genome assembly: hg19. Rat genome assembly: rn5.
Figure 4.
Figure 4.
Real-time PCR genotyping plots. X-axis show value of FAM probe signal (transgene specific probe). Y-axis show value of HEX probe signal (wild-type specific probe). RFU: relative fluorescence units. Each data point represents one animal. Cq values are means of values from two technical replicates. For each plot the tested genotypes are indicated in the legend. TG/TG: homozygous transgenic. WT/WT: wild-type. TG/WT: hemizygous transgenic. WT control: C57BL/6N. Neg. control: no template control.
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