The Cd6 gene as a permissive locus for targeted transgenesis in the mouse

Abstract

The introduction of a transgene into the genome through homologous recombination or sequence-specific enzymatic modification is a key technique for producing transgenic mice. The Rosa26 gene has been widely used to produce transgenic mice because the gene is transcriptionally active in various cell types and, at many developmental stages, is permissive for constitutive expression of integrated transgenes, and is dispensable for normal development. However, permissive loci other than Rosa26 are needed to generate mice that harbor multiple transgenes for complex studies. Here, we identified the Cd6 locus on mouse chromosome 19 as a transgene integration site in a transgenic mouse strain showing widespread reporter expression. Using this locus, we generated a knock-in mouse line that harbors a CAG promoter-driven reporter transgene, and found that the homozygous transgenic mice are viable and fertile, although transgene insertion disrupted Cd6 gene expression. The transgene on the Cd6 locus expressed reporter genes extensively throughout embryos, neonates, and adults. Combined with the Cre/loxP binary system, blood and lymphatic endothelial cell-specific reporter expression from the transgenic locus was achieved. These results suggest that Cd6 is valuable as an alternative site for targeted transgenesis.

Establishment of a tamoxifen-inducible Cre-driver mouse strain for widespread and temporal genetic modification in adult mice

“…To examine Cre recombination pattern and efficiency in more detail, we used CGE transgenic mice as a Cre reporter. CGE transgenic mice express EGFP after Cre-mediated excision of floxed cassettes under the control of the CAG promoter in the Cd6 locus [ 7 ] ( Suppl. Fig.…”

“…We purchased C57BL/6J and MCH:ICR mice from CLEA Japan (Tokyo, Japan); ROSA26-loxP-stop-loxP-β-geo knock-in mice (Gt(ROSA)26Sor tm1Sho ) [ 13 ] from the Jackson Laboratory (Bar Harbor, ME, USA); and FLP66 transgenic (RBRC01252) [ 23 ] and CAG-FLPe36 transgenic mice (RBRC01834) [ 9 ] from RIKEN BRC (Tsukuba, Japan). CGE transgenic mice and T26 transgenic mice were described previously [ 7 , 26 ]. All mice were housed under pathogen-free conditions.…”

“…GFP immunostaining of sections was performed as previously described [ 7 ]. Rat anti-GFP (Nacalai Tesque, Kyoto, Japan) was used as a primary antibody.…”

Gene Therapy for Cystic Fibrosis: Progress and Challenges of Genome Editing

“…Programmable nucleases offer the opportunity to precisely target integration of DNA sequences at their cleavage sites [40,41]. This represents a true advancement relative to conventional integration mediated by retroviral vectors associated with occasional insertional mutagenesis, epigenetic silencing and the altered expression levels driven by an exogenous promoter [39,42,43]. Homology-independent targeted integration (HITI) uses CRISPR-Cas mediated cleavages, both on target and incoming DNA, to catalyze the integration of DNA sequences at specific genomic sites and in the correct orientation [41].…”

Establishing a genetically engineered mouse ES cell line expressing an inducible Xist transgene along chromosome 19

“…This was to ensure that the gene of interest would not be silenced after insertion at the chosen site. The Cd6 gene was selected based on findings from a previously published paper which identified this locus as a transgene integration site that showed widespread reporter expression (Ichise et al, 2014). The Cd6 gene codes for CD6, a cell-surface receptor expressed on the majority of T-cells as well as a subset of B-cells in both humans and mice (Robinson et al, 1995).…”