The Kaestner lab is employing modern mouse genetic approaches, such as gene targeting, tissue-specific and inducible gene ablation, to understand the molecular mechanisms of organogenesis and physiology of the liver, pancreas and gastrointestinal tract.
Cdx2 null mice die before gastrulation. Cdx2loxP/loxP, however, are viable and fertile with a fuctionally wild type Cdx2loxP allele.
A conditional Cdx2 allele was derived by targeting embryonic stem cells. After germ line transmission of the targeted allele, the FRT-flanked neomycin resistance gene was removed by crossing to Flp1 deleter mice. Cdx2loxP/loxP were derived by intercrossing Cdx2loxP/+ mice.
These mice can be used to develop conditional Cdx2 knockouts.
Dicer1loxP/loxP conditional mutant mice (C57BL6) were crossed with Villin-Cre transgenic mice.
These mice can be used to generate tissue-specific Foxa1 and Foxa2 knockouts.
Transgenic lines were obtained by pronuclear injection into fertilized C57BL/6;SJLF1 hybrid oocytes. This line displays efficient deletion of Foxa2 in the liver.
A transgenic founder with integration of the full Foxa3/Cdx2 YAC was derived from Foxa3/Cdx2 YAC-injected oocytes. Cdx2 is expressed early in fetal development and continues to be expressed in the gastric mucosa throughout development and adulthood. These mice express Cdx2 protein throughout the epithelium of the glandular gastric mucosa. The ectopic expression of Cdx2 in the gastric epithelium is sufficient to cause transdifferentiation of the gastric mucosa to an intestinal cell-type characteristic of human intestinal metaplasia.
These transgenic mice express Cre recombinase under the control of the mouse Foxl1 promoter for the deletion of loxP flanked sequences in the gastrointestinal tract. Foxl1-Cre activity is confined to a few mesenchmyal cell layers adjacent to the endoderm-derived gut epithelium.
Foxl1-Cre line was crossed to Rosa26-YFP reporter mice in which the yellow fluorescent protein (YFP) is expressed only after Cre-mediated excision of a loxP-flanked stop codon.
The Foxl1-Cre; RosaYFP model allows for easy isolation of adult hepatic progenitor cells that can be expanded and differentiated in culture. For cell replacement therapy, it is critical to isolate hepatic progenitor cells, expand them in culture, and characterize their biology.
A series of crosses was used to produce mice with intestine-specific deletion of Foxa1 and Foxa2. Foxa1 and Foxa2 proteins were absent from the small intestine, colon, and rectum.
The expression of Cre recombinase in AlfpCre mice is driven by the Alb promoter and both Alb and Afp enhancers. Both Afp and Alb are initially expressed in the hepatoblast at E9.0. Ablation of Foxa1/2 was first apparent in the liver at E16.5, complete by P2, and maintained into adulthood.
These mice were generated by breeding Foxa2loxP/loxP homozygous mice with Ins.Cre transgenic mice, which express the Cre recombinase cDNA under the control of the beta cell-specific rat insulin 2 promoter. The resulting Foxa2loxP/+; Ins.Cre offspring were mated to Foxa2loxP/loxP homozygotes to obtain the Foxa2loxP/loxP; Ins.Cre mice.
The Cre coding sequence was fused to a 2 kb fragment of the mouse albumin promoter and a 4.6 kb fragment of the mouse alpha-feto protein enhancer.
Cdx2 complementary DNA was introduced into a 170-kilobase (kb) Foxa3-YAC.