Identification and survival analysis of TTA/TETO-CCKR-2 double transgenic mice / 中国组织工程研究

ABSTRACT

BACKGROUND: The inducible forebrain-specific cholecystokinin receptor-2 (CCKR-2) double transgenic (tTA/tetO-CCKR-2 tg, abbreviated as dtg) mice are an ideal model of anxiety-related diseases. However, there is still a lack of model identification and life related data OBJECTIVE: To identify the genomic DNA of the offspring and the specific expression of CCKR-2 transgene in the forebrain, and to analyze the survival probability of dtg mice. METHODS: α-CaMKII/tTA single transgenic mice and tetO-CCKR-2 single transgenic mice were cross-fertilized to construct a dtg mouse model. The genomic DNA was extracted from the tail of the offspring, and the genotypes were detected by PCR and agarose gel electrophoresis. Wild-type (WT) mice were used as controls. In situ hybridization was used to detect the expression of CCKR-2. Survival of dtg mice and WT mice (30 females and 30 males) was observed and recorded within 2 years. The study protocol was approved by the Experimental Animal Ethics Committee of Southwest Medical University, with an approval No. 20150068. RESULTS AND CONCLUSION: Agarose gel electrophoresis results showed the molecular weight of the PCR products of dtg mice was consistent with the expected target gene fragment. In situ hybridization results showed a strong signal of CCKR-2 was detected in the forebrain of dtg mice, but hardly present in the WT mice. The median survival time of dtg mice was 76 weeks in females and 77 weeks in males. The survival probability was decreased with age in dtg mice. The survival probability of WT mice was significantly better than that of dtg mice (P < 0.001). There was no significant sex difference between males and females of dtg mice (P=0.577). Therefore, the specific expression of CCKR-2 transgene in the forebrain can be identified using PCR amplification, genomic DNA extraction, agarose gel electrophoresis, and in situ hybridization. tTA/tetO-CCKR-2 double transgenic induction may shorten the survival time of mice, but no significant difference is observed between the females and males of dtg mice.