鼠牙乳头细胞核心结合因子cDNA片段的克隆与序列测定

Cloning and sequencing of rat cbfa1 cDNA fragment from dental papilla cell

目的 从培养的大鼠牙乳头细胞中克隆核心结合因子 (cbfa1)的cDNA片段。方法 原代培养大鼠牙乳头细胞 ,提取培养细胞的总RNA ,逆转录合成cDNA ,用PCR的方法扩增cbfa1基因片段。将获得的预期大小的PCR产物插入pUC18克隆载体中 ,转化TOP10感受态细胞 ,蓝白筛选白色克隆 ,进行体外扩增 ,提取质粒进行酶切鉴定 ,含目的插段的克隆在PE317 A自动测序仪上进行序列测定。结果 从培养的鼠牙乳头细胞提取的总RNA中成功克隆出 6 91bpcbfa1基因片段 ,测序结果和GenBank报道序列完全一致。结论 从鼠牙乳头细胞中成功克隆到cbfa1基因片段 ,确切的证实了核心结合因子 (cbfa1)基因在鼠牙乳头细胞中的表达。

Objective To clone the complementary cDNA fragment of cbfa1 from dental papilla cell of rat. Methods Total RNA was extracted from primary culture of rat dental papilla cells and subsequently reversely transcripted into cDNA followed by polymerase chain reaction (PCR) to trap the cDNA fragment of interest. The PCR product of expected size was then inserted into cloning vector pUC19a and transformed into TOP10 host strains for amplification. Positive recombinant clones were screened out by white blue selection and restriction analysis for sequencing in PE317 A automatic DNA sequencer. Results A 691bp cDNA fragment was cloned from the total RNA of rat dental papilla cell and proved to be identical to the rat cbfa1 sequence recorded in GenBank. Conclusion Rat cbfa1 cDNA fragment is successfully cloned from rat dental papilla cells and proues the expression of cbfa1 gene in rat dental papilla cells.