Establishment Of CaMKⅡα-Cre Transgenic Mice Model

Conditional gene knockout mediated by Cre-LoxP system is one of the most important technologies for study of gene function. It overcomes some drawbacks of conventional gene knockout technology. In the study of the nervous system, this technology was used to made it possible to limit the gene knockout in a sub-region of the brain or a certain stage during the development, which facilitates the detailed study of gene in brain. Choosing a tissue-specific promoter is the most important for the conditional gene knockout in nervous system. The studies show that CaMKⅡαgene expression has strong temporal and spatial specificity. Therefore, the promoter of CaMKⅡαgene is suitable to generate the transgenic mouse in which Cre recombinase expression occurs in the forebrain specifically.The neuron-specific expression vector with Cre recombinase (PCCEⅠ) was previously constructed in our laboratory. In this experiment, the linearized PCCEⅠvector was introduced into fertilized eggs by pronuclear microinjection to obtain the CaMKⅡα-Cre transgenic mice. Then we tested the expression of Cre gene in the transgenic mice. This study lays a foundation for the generation of the conditional ADAM10 gene knockout mice, which will help to understand the pathogenesis of Alzheimer's disease.12.318 kb DNA fragments of CaMKⅡα-Cre transgene were introduced into fertilized eggs by microinjection. 81 offsprings were obtained. Seven founder mice( i.e. PCCE5,PCCE10, PCCE19, PCCE59, PCCE60, PCCE65 and PCCE75) carrying the CaMKⅡα-Cre transgene were identified by genomic PCR. The integration efficiency is 8.6%. The CaMKⅡα-Cre transgenic mice were bred with C57BL/6J mice, the positive transgenic mice were screened by genomic PCR. The results of RT-PCR testing show that Cre genes were only expressed in the cortex and hippocampus of PCCE10, PCCE 19 and PCCE 60 transgenic mice, while not expressed in the other tissues. The CaMKⅡα-Cre transgenic mice were bred with ADAM10~LoxP/LoxP transgenic mice. The results showed that the deletion of Exon 3 in ADAM10 gene occurred in the cortex and hippocampus of the resulting ADAM10~+/–/ Cre mice. The CaMKⅡα-Cre transgenic mice were bred with Rosa26-LacZ mice to detect the tissue distribution and the activity of Cre recombinase in the transgenic mice. The results of LacZ staining in the ROSA26~ LacZ/ LacZ/ Cre double transgenic mice reveals that LacZ gene were only expressed in the brain (especially the forebrain and hippocampus) at day P28 , while sparsely expressed in a few cells of the forebrain cortex and hippocampus at day P1. LacZ gene expression was not found in brain at day E16, demonstrating that Cre recombinases play a role in the postnatal brain. Finally, the transgene copy numbers in transgenic mice were determined by real-time fluorescent quantitative PCR. Three CaMKⅡα-Cre founders ( i.e. PCCE10, PCCE19 and PCCE60) were detected to have the transgene copies of 19, 7 and 5, respectively. The homozygous mice were obtained by this method and confirmed by genetic breeding.In summary, We have generated three CaMKⅡα-Cre transgenic mice in which Cre recombinase gene were specifically expressed in the forebrain and successfully mediated the knockout of the floxed target gene. These homozygous CaMKⅡα-Cre transgenic mice could serve as a valuable tool for studying the associated gene function and diseases of the nervous system.