摘要
该文旨在鉴定特异敲低Ⅱ型肺泡上皮细胞中Brg1(Brahma-related gene 1)基因小鼠的基因型,为进一步研究Brg1在肺相关疾病中的作用奠定基础。将两对转基因小鼠Brg1fl/fl和SP-C-rt TA/(tet O)7-Cre进行繁殖交配,将得到纯合子、杂合子及野生型3种基因型。出生后1周龄剪尾提取基因组DNA,PCR法判定基因型。用多西环素诱导法诱导(tet O)7-Cre重组酶活性,靶向剪切Ⅱ型肺泡上皮细胞上Brg1基因,磁珠分选法提取小鼠原代Ⅱ型肺泡上皮细胞,通过观察细胞生长特征、检测pro SP-C表达鉴定原代Ⅱ型肺泡上皮细胞,RT-PCR法、Western blot检测Brg1表达水平。结果显示,成功繁殖了Brg1纯合子小鼠,通过磁珠分选法成功分离了原代Ⅱ型肺泡上皮细胞,用多西环素诱导法成功敲低了Ⅱ型肺泡上皮细胞Brg1基因。该结果为相关研究提供动物模型奠定了基础。
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/fland SP-C-rt TA/（tet O）7-Cre, were introduced to breed and mated, and homozygous（SP-C-rt TA/（tet O）7-Cre/Brg1fl/fl）, heterozygous（SP-Crt TA/（tet O）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 AEC2 s after 7 days Doxycycline-inducible. The phenotype of isolated and cultured AEC2 s was confirmed by observation of the morphology of AEC2 s and detection of the expression of prosurfactant protein-C（pro SP-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 AEC2 s were successfully extracted, and the specific knocking down of Brg1 in AEC2 s was successful. This study provided the animal experimental model for the relevant research.
出处
《中国细胞生物学学报》
CAS
CSCD
2017年第3期280-287,共8页
Chinese Journal of Cell Biology
基金
重庆市科技委员会重大专项(批准号:Cstc2014yykfC10003)资助的课题~~
