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

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.

Mapping Transgene Insertion Sites Reveals Complex Interactions Between Mouse Transgenes and Neighboring Endogenous Genes

“…Targeted Locus Amplification (TLA) technology uses the physical proximity of nucleotides within a locus of interest to generate a map of original sequences and corresponding inserted transgenes (de Vree et al, 2014). Transgenic homozygotes and wild-type controls were euthanized and cells were prepared from their spleens (Cain Hom et al, 2017). Homozygotes were distinguished from heterozygotes by fluorescent quantitative PCR (qPCR) results from a commercial genotyping service (Transnetyx; http://www.transnetyx.com/).…”

“…al, 1999;Suzuki et al, 2006;Sha et al, 2007;Liang et al, 2008;Dubose et al, 2013;Srivastava et al, 2014;Raman et al, 2015) have been cumbersome, little used and, in our hands, largely unsuccessful. Recently, however, a newly developed method termed Targeted Locus Amplification (TLA) was introduced that appeared to be more promising (de Vree et al, 2014;Cain-Hom et al, 2017). In TLA, genomic DNA in nuclei is cross-linked by formaldehyde, digested into small fragments by the frequently cutting NlaIII restriction enzyme and religated to form larger circular DNA containing fragments that were likely to have been near neighbors on a chromosome.…”

Enhanced CHO Clone Screening: Application of Targeted Locus Amplification and Next‐Generation Sequencing Technologies for Cell Line Development

“…As such, TLA allows for the targeted sequencing of loci of interest and detection of single‐nucleotide variations (SNVs) and structural variants . TLA has previously been shown to have important advantages in the targeted complete NGS of transgenes and transgene integration sites …”

Locating a transgene integration site by nanopore sequencing

“…Current methods to identify sites of integration are labor intensive or require the design of specialized assays and computational expertise (Itoh et al 2015;Cain-Hom et al 2017). For these reasons, only 433 of the 8,715 transgenic alleles reported in the mouse genome database are currently annotated with a chromosomal location (www.informatics.jax.org/).…”