The Cre-Lox system allows for gene knockout/knockin in specific cells/tissues, with the timing of its expression is determined by the promoter. To further achieve dual control of gene expression in terms of both time and space, ligand-dependent CreER recombinase was developed. CreER is composed of Cre recombinase fused with the hormone-binding domain of the estrogen receptor. In the absence of the estrogen analog tamoxifen, the recombinase is mainly present in the cytoplasm. Only under the action of tamoxifen can the recombinase enter the cell nucleus and exert its recombination function. CreERT2 is an upgraded version of the CreER recombinase with three point mutations (G400V/M543A/L544A) in the estrogen-binding domain, which reduces background activity when uninduced while increasing tamoxifen sensitivity and induction efficiency. Today, we introduce the improved pancreas-specific cre driver mouse line — the Pdx1-CreERT2 mouse model (Product No.: C001537).
PDX1 Gene: A Key to Pancreatic Organogenesis and β-cell Function
The PDX1 gene encodes a protein that is a major regulator of pancreatic organogenesis, β-cell maturation and maintenance, and normal insulin function. Activation of the PDX1 gene promotes insulin release and the expression of important genes in β-cells, making it necessary for pancreatic stem cells to differentiate into pancreatic β-cells. Thus, PDX1 is an important target for gene or replacement therapies for diabetes.[1]
Research has found that PDX1 protein maintains the characteristics and functions of β-cells by inhibiting the differentiation of α-cells, and pancreatic β-cells can survive and undergo β-to-α cell reprogramming in the absence of PDX1.[2] PDX1 is specifically expressed in early pancreatic epithelium and plays a role in proliferation and differentiation during development. At the adult stage, PDX1 is essential for hormone production in β-cells. PDX1 is one of the earliest expressed transcription factors during pancreatic development and continues to be expressed during β-cell maturation.[1,3] Besides its expression in β-cells and some δ-cells, it is also expressed in the gastrointestinal tract (such as the duodenum) and the central nervous system during development.[4-5]
Figure 1. The role of PDX1 in pancreatic organogenesis and β-cell maturation.[1]
Pdx1-CreERT2 Mouse Model for Targeted Pancreatic Research
Cyagen has independently developed the Pdx1-CreERT2 mouse (Product No.: C001537) using gene editing technology to provide a liver-specific Cre driver mouse line with exceptional temporal control for gene research and preclinical studies. The Pdx1-CreERT2 mouse model expresses the CreERT2 recombinase under the control of the mouse Pdx1 gene regulatory elements. Once Pdx1-CreERT2 mice are crossed with mice containing loxP sites, tamoxifen induction can trigger Cre recombinase-mediated recombination between the loxP sites in the pancreas of the offspring.
Tamoxifen Induction of Pancreas Tissue-Specific Gene Recombination
Without tamoxifen treatment, CreERT2 recombinase primarily resides in the cytoplasm. Only when tamoxifen is administered does the CreERT2 recombinase enter the cell nucleus and exert its recombination activity. When Pdx1-CreERT2 mice are crossed with mice containing loxP sites, tamoxifen induction can trigger sequence recombination between loxP sites mediated by Cre recombinase in the pancreatic cells of the offspring. It’s important to note that without tamoxifen treatment, there may be some leakage of CreERT2 recombinase expression before induction. The insertion site of the Cre recombinase gene expression cassette in this strain is located on chromosome 5, so breeding with gene-edited mice targeting genes on the same chromosome as the Cre mouse should be avoided when conducting mating.
Pdx1-CreERT2 Mice Exhibit Higher Pancreas-Specific Recombination Efficiency than Pdx1-Cre Mice
Figure 2. Expression of Cre recombinase in the pancreas shown by fluorescence microscopy.
After Pdx1-CreERT2 mice are crossed with ROSA26-LSL-tdTomato mice, which conditionally express tdTomato fluorescent protein, the expression of CreERT2 recombinase is induced in the double transgenic offspring by treatment with tamoxifen or corn oil. The fluorescence microscopy results show a significant amount of tdTomato fluorescence signal in the pancreas of the tamoxifen-treated group, indicating high recombinase activity. Additionally, compared to non-inducible Pdx1-Cre mice, the Pdx1-CreERT2 mice exhibit significantly higher recombination efficiency in the pancreas.
Cre Recombinase Exhibits Limited Expression in the Duodenum and Thymus
Figure 3. Fluorescence microscopy demonstrating the expression of Cre recombinase in the duodenum and thymus.
Following the previous method of crossing Pdx1-CreERT2 mice with ROSA26-LSL-tdTomato mice, the expression of CreERT2 recombinase is induced in the offspring by treatment with tamoxifen or corn oil. The fluorescence microscopy results show partial red fluorescence signals in the duodenal villi and thymus of the tamoxifen-treated group, demonstrating additional tissues that may be affected by the induction of gene modification.
The Pdx1-CreERT2 Mouse Model Exhibits Good Tissue Specificity
Figure 4. Immunofluorescence (IF) shows no demonstrable expression of Cre recombinase in other tissues tested, including the stomach, uterus, ovaries, lungs, liver, and brain.
When Pdx1-CreERT2 mice are crossed with ROSA26-LSL-tdTomato mice and their offspring are treated with tamoxifen or corn oil, no significant recombination signals are detected in the stomach, uterus, ovaries, lungs, liver, and brain tissues of either the tamoxifen-treated group or the corn oil-treated group. This indicates that the Pdx1-CreERT2 mouse exhibits good tissue specificity, with high targeting of the pancreas and detargeting of other major tissues.
Summary
The Pdx1-CreERT2 mouse model (Product No.: C001537) has demonstrated high expression of Cre recombinase in the pancreas, with minimal fluorescence signals observed in the duodenum and thymus indicating trace recombination events. No recombination signals are detected under fluorescence microscopy in the stomach, uterus, ovaries, lungs, liver, and brain — indicating detargeting of other major tissues. Therefore, the Pdx1-CreERT2 mouse model demonstrates good specificity for use in targeted genetic research on pancreatic islet cell tissues.
>> Learn More: Pdx1-CreERT2 Mouse Model
References:
[1]Ebrahim N, Shakirova K, Dashinimaev E. PDX1 is the cornerstone of pancreatic β-cell functions and identity. Front Mol Biosci. 2022 Dec 15;9:1091757.
[2]Gao T, McKenna B, Li C, Reichert M, Nguyen J, Singh T, Yang C, Pannikar A, Doliba N, Zhang T, Stoffers DA, Edlund H, Matschinsky F, Stein R, Stanger BZ. Pdx1 maintains β cell identity and function by repressing an α cell program. Cell Metab. 2014 Feb 4;19(2):259-71.
[3]Jennings RE, Berry AA, Kirkwood-Wilson R, Roberts NA, Hearn T, Salisbury RJ, Blaylock J, Piper Hanley K, Hanley NA. Development of the human pancreas from foregut to endocrine commitment. Diabetes. 2013 Oct;62(10):3514-22.
[4]Ma J, Chen M, Wang J, Xia HH, Zhu S, Liang Y, Gu Q, Qiao L, Dai Y, Zou B, Li Z, Zhang Y, Lan H, Wong BC. Pancreatic duodenal homeobox-1 (PDX1) functions as a tumor suppressor in gastric cancer. Carcinogenesis. 2008 Jul;29(7):1327-33.
[5]Perez-Villamil B, Schwartz PT, Vallejo M. The pancreatic homeodomain transcription factor IDX1/IPF1 is expressed in neural cells during brain development. Endocrinology. 1999 Aug;140(8):3857-60.
