| The gastric epithelium cells are highly turnover, different cells have different refresh cycles, and the whole epithelium is in a dynamic and delicate balance, the breakout of this balance leads to various diseases happened. The gastric mucosa is composed of lots of gastric unit, each unit is composed of pit cells, parietal cells, chief cells, mucous neck cells, multipotent stem cells and minor enteroendocrine cells, all these cells are derived from the mutipotent stem cells which located at the isthmus regions. Different cells have different functions and different refresh cycles, the changes of the functions and refresh cycles of the gastric epithelium cells effect the stabilization of the gastric mucosa directly. The disorders of differentiation, proliferation and apoptosis of the epithelium cells are the reasons of kinds of gastric diseases.Pit cells are localized to the edge of the gastric mucosa, serving as the first protective barrier of the mucosa. They secrete mucus to protect the gastric epithelium from high salt, acid and pepsin. Mucus-producing pit cells are derived from the stem cells located at the isthmus, they turn over every 3 days. Parietal cells are highly differentiated, located at the isthum and the body of the gastric mucosa gland. The main function of parietal cells is secreting acid. The turnover time of parietal cells is about 54 days, they composed about 30% of the gastric epithelium cells, and they are the largest and most cells of the gland. Chief cells, also named as zymogenic cells are located at the bottom of the gland,they turn over every 190 days. Chief cells secrete pepsinogen to the lumen and activated by the acid secreted by parietal cells. These three cell lineages are the principal cells of the gastric gland.The Cre-loxP system provides a powerful means of studying genes function in specific tissues and specific cells. But, the gastric epithielium cells cpecific Cre transgenic mice is still very rare, and this hampered us to beter understand the development of the gastric, and also hampered us to study the mechanism of gastric diseases. To better understand the function of gastric epithelium cells in the homeostasis maintenance of the gastric mucosa, also to better understand their functions in the progression of gastric diseases, we established three transgenic mouse lines in which the Cre recombinase were expressed in pit cells, parietal cells and chief cells, respectively.The use of tissue specific promoter is the key of establishment of Cre transgenic mice. Calpains are Ca2+-regulated cysteine proteases. Mammals have 14 calpain genes, of which 7 are predominantly expressed in specific organs. NCL-2/calpain-8 (Capn8) has been identified as a stomach-specific calpain, whose expression is strictly localized at gastric pit cells.β-subunit of H+, K+-ATPase(Atp4b) is reported to be a parietal cells marker. Pepsinogen C (Pgc) was expressed specially in gastric chief cells, and was used as a chief cell marker. So, we used the promoters of Capn8, Atp4b and Pgc to drive the expression of Cre recombinase specially in pit cells, parietal cells and chief cells, respectively.The 3.9-kb promoter of Capn8 gene and 2.1-kb promoter of Pgc gene and 1.0-kb promoter of Atp4b gene were obtained from mouse genomic DNA by PCR amplification. The PCR products were inserted into the cloning sites of an engineered vector containing Cre coding region and hGH polyadenylation signal. The final transgenic constructs consisted of 5′sequence of promoter regions and 1.2-kb of the Cre coding region followed by the 2.1-kb of hGH polyadenylation signal. After linearized, the inserts were microinjected into the pronuclei of fertilized FVB mouse oocytes to generate the transgenic mice. Identifying the offsprings of the transgenic mice, we established three transgenic mouse lines: Capn8-Cre, Atp4b-Cre and Pgc-Cre.To check the tissue distribution and the excision activity of the Cre recombinase, the transgenic mice were crossed with a mouse strain that carries the Smad4 conditional alleles (Smad4Co/Co). The Cre-mediated excision of exon 8 of Smad4 in different tissues of the Capn8-Cre;Smad4Co/+offsprings were evaluated by PCR. Genomic DNAs were extracted from different tissues, and PCR analysis revealed Cre-mediated recombination in the stomach of all the three transgenic mice. In the Capn8-Cre;Smad4Co/+ mouse, the Cre mediated recombination also can be detected in the liver and skin tissues. To identify the exact cell types in which Cre recombinase performs its excision function, we bred the Capn8-Cre transgenic mouse with the reporter mouse ROSA26. LacZ activity was detected specially in the pit cells of the Capn8-Cre;ROSA26 double transgenic mice, In addition, Cre activity was also detected in a small subpopulation of keratinocytes located in the interfollicular epidermis of the skin and hepatocytes of the Capn8-Cre;ROSA26 transgenic mice. Furthermore, imunohistochemical staining of Smad4 in the corpus region of the Capn8-Cre;Smad4Co/Cotransgenic mouse, showed that the expression of Smad4 was significantly decreased in the pit cells.Using the same methord we identified the tissues that expressed the Cre recombinase of Pgc-Cre and Atp4b-Cre mouse. In the Pgc-Cre mouse the Cre mediated recombination can be detected in the gastric chief cells and duodenum. In the Atp4b-Cre transgenic mouse, the Cre-mediated recombination only can be detected in the gastric parietal cells.To verifyication the efficiency of the Cre recombinase of Capn8-Cre and Pgc-Cre mice, we bred these transgenic mice with a mouse strain that carries the Smad4 conditional alleles (Smad4Co/Co), and we got pit cells and chief cells specific Smad4 knockout mice: Capn8-Cre;Smad4Co/Co and Pgc-Cre;Smad4Co/Co. There are some histological differences was observed between the Smad4 knockout mouse and the control mouse.In conclusion, we established three transgenic mouse lines: Capn8-Cre, Atp4b-Cre and Pgc-Cre, using 3.9-kb promoter of Capn8 gene, 1.0-kb promoter of Atp4b gene and 2.1-kb promoter of Pgc gene, respectively. The Cre recombinase of the three transgenic mouse line can mediated efficient recombination specially in gastric pit cells, parietal cells and chief cells. The three transgenic mouse lines provide us useful tools for study the mechanism of the differentiation and genetic control of gastric epithelium cells. |
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