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Genotype Identification of Mice with Conditional Brg1 Knocking down in Type II Alveolar Epithelial Cells
Si Daozhu1, Peng Danyi1, Zhang Rong3, Xia Yunqiu2, Niu Chao2, Gou Hao1, Liu Jiang1, Wang Lijia1, Tian Daiyin2, Liu Enmei2, Zou Lin1, Fu Zhou1*
1Department of Pediatric Research Institute, Children’s Hospital of Chongqing Medical University, Ministry of Education Key Laboratory of Child Development and Disorders, China International Science and Technology ooperation Base of Child Development and Critical Disorders, Chongqing Engineering Research Center of Stem Cell Therapy, Chongqing 400014,China;
2Respiratory Center of Children’s Hospital of Chongqing Medical University, Chongqing 400014, China;
3Xijing Hospital, The First Affiliated Hospital of the Fourth Military Medical University, Xi’an 710000, China
2Respiratory Center of Children’s Hospital of Chongqing Medical University, Chongqing 400014, China;
3Xijing Hospital, The First Affiliated Hospital of the Fourth Military Medical University, Xi’an 710000, China
Abstract: The purpose of this study was to identify the genotype of knocking down of Brg1 (Brahmarelated gene1) gene in type II alveolar epithelial cells (AEC2s) in mice， and to lay a foundation for further study on the role of Brg1 in lung related diseases. Two pairs of transgenic mice， Brg1fl/fl and SP-C-rtTA/(tetO)7-Cre，were introduced to breed and mated， and homozygous (SP-C-rtTA/(tetO)7-Cre/Brg1fl/fl)， heterozygous (SP-CrtTA/(tetO)7-Cre/Brg1fl/–) and wild-type genotypes were obtained. Genomic DNA was extracted from the tail of one-week-old mice. PCR/Q-PCR were used to identify the genotype. Gentle MACS Dissociator was applied for isolation and purification of primary AEC2s after 7 days Doxycycline-inducible. The phenotype of isolated and cultured AEC2s was confirmed by observation of the morphology of AEC2s and detection of the expression of prosurfactant protein-C (proSP-C). The success of Brg1-knockdown was measured by RT-PCR and Western blot.The results showed that the feeding and breeding of Brg1 homozygous mice were successful. Primary AEC2s were successfully extracted， and the specific knocking down of Brg1 in AEC2s was successful. This study provided the animal experimental model for the relevant research.
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