Reproduction： Carrier x WT
The Nppa gene encodes a protein that belongs to the natriuretic peptide family, which is involved in controlling extracellular fluid volume and electrolyte homeostasis. The protein first forms a precursor containing a signal peptide, which is processed to release a peptide similar to vasoactive peptides and cardiac diastolic peptides from the N-terminus and a peptide with natriuretic activity from the C-terminus. Mutations in the Nppa gene are associated with familial atrial fibrillation 6. In prenatal mice, the Nppa gene is first expressed in the ventricles and subsequently shifts to expression in the atria. In the hearts of healthy postnatal female mice, Nppa expression is limited to the atria as part of the fetal gene activation program. In adult mice, Nppa can be re-expressed in the ventricles in response to pathological stress. The Nppa gene can serve as an important marker for studying cardiac chamber formation and cardiac specification, as well as for investigating the molecular mechanisms of myocardial hypertrophy and heart failure due to its unique expression pattern.
This strain was constructed using gene editing technology. Part of the exon 1 coding sequence was replaced with the “Cre-rBG pA” cassette. Nppa-Cre mice can specifically express Cre recombinase in ventricular and atrial cardiomyocytes. When this strain is crossed with mice containing loxP sites, the offspring can produce sequence recombination between loxP sites mediated by Cre recombinase in cardiomyocytes. The heterozygous Nppa-Cre mice are viable and fertile.
Part of the exon 1 coding sequence was replaced with the “Cre-rBG pA” cassette.
The Nppa-Cre mice were bred with Rosa26-LSL-tdTomato mice to generate double heterozygous offspring. Cre-mediated recombination will result in the expression of tdTomato protein in the Cre-positive cells of the offspring. Tissues such as the heart, brain, testis, uterus, colon, spleen, and lungs were collected from 6-week-old offspring mice. The immunohistochemistry and immunofluorescence staining were used to observe the expression of tdTomato protein in different tissues to determine the activity of Cre recombinase.
1. Immunohistochemistry of the cardiac tissue
Figure 1. Immunohistochemistry of the cardiac tissue. Cardiac tissues from 6-week-old offspring mice were collected and immunohistochemistry was used to observe the recombination. The results showed that, compared to the control group (Cre- mice), there was a significant tdTomato signal in the cardiomyocytes of Cre+ mice, indicating that recombination occurred in the cardiomyocytes of Cre+ mice.
2. Expression of Cre recombinase in the cardiac tissues
Figure 2. Immunofluorescence staining of the cardiac tissue. The atrial and ventricular cardiomyocytes and vascular endothelial cells of Cre+ mice showed significant red fluorescence of tdTomato protein, indicating the presence of Cre recombinase expression.
3. Expression of Cre recombinase in the large intestine, lungs, brain, and spleen
Figure 3. Immunofluorescence staining of the large intestine, lungs, brain, and spleen. Red fluorescence from tdTomato protein was barely observable in the large intestine of the Cre+ mice. The lung smooth muscle and brain (mainly in the hippocampus and cortex) of the Cre+ mice showed a red fluorescence signal. A small amount of red fluorescence was also observed in the spleen of Cre+ mice. In summary, Cre+ mice showed almost no expression of Cre recombinase in the large intestine, while Cre recombinase expression was present in some cells of the lung, brain, and spleen. In contrast, red fluorescence was undetectable in Cre- mice.
4. Expression of Cre recombinase in the uterus and testes
Figure 4. Immunofluorescence staining of uterus and testes. The uterus of the Cre+ mice showed a small amount of red fluorescence signal. Red fluorescence was present in some spermatogenic regions. A small amount of fluorescence was observed in the testicular mesenchyme and secondary spermatocytes, indicating that partial recombination occurred in the testicular mesenchyme and secondary spermatocytes. In summary, Cre recombinase expression was present in some cells of the uterus and testes. In contrast, red fluorescence was undetectable in Cre- mice.
In Nppa-Cre mice, Cre recombinase expression is primarily localized to the atrial and ventricular cardiomyocytes and vascular endothelial cells, while Cre recombination can occur in some cells of the lung, brain, spleen, uterus, and testis. In contrast, Cre recombinase expression was almost absent in the large intestine. In summary, the Nppa-Cre mice were constructed successfully. However, using male mice to breed with Nppa-Cre mice may have reproductive leakage.
The Cre recombinase gene is located on chromosome 4 in Nppa-Cre mice. Please avoid using mice that have been targeted on the same chromosome for breeding.
 Irina A. Sergeeva, Ingeborg B. Hooijkaas, Ingeborg Van Der Made, Willeke M.C. Jong, Esther E. Creemers, Vincent M. Christoffels, A transgenic mouse model for the simultaneous monitoring of ANF and BNP gene activity during heart development and disease, Cardiovascular Research, Volume 101, Issue 1, 1 January 2014, Pages 78–86.