Generating somatic mosaicism with a Cre recombinase-microsatellite sequence transgene

Abstract

Strategies for altering constitutional or somatic genotype in mice are well established, but approaches to generate mosaic genotypes in mouse tissues are limited. We showed that a functionally inactive Cre recombinase transgene with a long mononucleotide tract altering the reading frame was stochastically activated in the mouse intestinal tract. We demonstrated the utility of this approach by inducing colonic polyposis after Cre-mediated bi-allelic inactivation of the Apc gene.

Figure 1

CDX2P9.5-G22Cre transgenic mice and somatic activation of Cre function. (a) Schematic representation of CDX2P9.5-G22Cre transgene construct. (b) A CDX2P9.5-G22Cre transgenic embryo (17.5 d post-coitum) from a homozygous R26R reporter female mouse crossed with an F1 CDX2P9.5-G22Cre transgenic male (line 189). Specific β-gal staining was seen only on the tip of the tail (top right) as compared with the absence of β-gal staining in wild-type embryo (bottom right). Scale bars, 2 mm. (c) X-gal staining of gastrointestinal tract tissues from a 6-week-old CDX2P9.5-G22Cre;R26R mouse. Stomach, followed by small intestine, cecum and colon, with anus at lower right (left panel). Stereomicroscope images of β-gal staining, with arrows indicating the cecal, proximal colon and distal colon regions visualized (middle). Cryostat sections of tissue from cecum, proximal colon and distal colon stained with X-gal (right). Scale bars, 1 cm (left), 1 mm (middle) and 100 μm (right). (d) Results of a quantitative β-gal assay of tissues comparing β-gal enzymatic activity in tissues from a 4-month-old CDX2P9.5-G22Cre;R26R mouse versus a control R26R mouse.

Figure 2

Polyposis in cecum and proximal colon in CDX2P9.5-G22Cre;Apc ^flox/flox mice. (a) Survival rates in CDX2P9.5-G22Cre;Apc ^flox/flox mice (red; n = 10) compared to CDX2P9.5-G22Cre;Apc^flox/+ mice (black; n = 6). (b) Dissected whole gastrointestinal tract of CDX2P9.5-G22Cre;Apc ^flox/flox mouse with extensive polyposis in cecum and proximal colon (left), with a higher power view of proximal colon polyposis (right). Scale bars, 1 cm (left) and 5 mm (right). (c) Histological analysis of hematoxylin and eosin–stained section from proximal colon of a CDX2P9.5-G22Cre;Apc ^flox/flox mouse, with tubular adenomatous glands replacing normal mucosa, and higher power hematoxylin and eosin–stained region of adenomatous glands (top right). Analysis of nuclear β-catenin staining in neoplastic cells (bottom right). Scale bars, 200 μm (left) and 25 μm (right).