Foxp1基因编码区序列的克隆及其在星形胶质细胞中的表达

Cloning of Foxp1 Coding Sequence and Its Expression in Astrocyte

目的体外分离培养原代星形胶质细胞,在星形胶质细胞中克隆Foxp1基因编码区序列(CDS)区并过表达。方法利用机械分离法获取小鼠原代星形胶质细胞;异硫氰酸胍法(TRIzol法)提取原代星形胶质细胞内总RNA并逆转录为cDNA,克隆CDS区、测序;利用美国国家生物技术信息中心(NCBI)和SnapGene软件对测序结果与NCBI库内小鼠中4个Foxp1转录本的CDS区进行对比;将星形胶质细胞中Foxp1基因的CDS克隆到表达载体上,并转染到HEK293T细胞内验证其表达。结果体外成功分离培养原代星形胶质细胞,且纯度达95%及以上;克隆出星形胶质细胞内Foxp1的CDS,相较参考序列Foxp1转录本4的CDS,其在514/515处插入3个连续碱基AGC;将克隆有Foxp1基因CDS区的表达载体转入HEK293T细胞内后可见在约106 kD处有Foxp1蛋白表达。结论成功分离培养原代星形胶质细胞并获取Foxp1新的CDS。

Objective To isolate and culture primary astrocytes in vitro,clone coding sequence(CDS)region of Foxp1 in primary astrocytes and overexpress it.Methods Mice primary astrocytes were isolated by mechanical separation method.Total RNA in primary astrocytes was extracted by Guanidine isothiocyanate(TRIzol)and reverse transcripted to cDNA,CDS of Foxp1 was cloned and sequenced.The website National Center of Biotechnology Information(NCBI)and software SnapGene were used to compare the sequencing results with the CDS of the forth Foxp1 transcripts in the mouse library of NCBI.Foxp1 overexpression vector was constructed by using the sequence of Foxp1 CDS cloned from astrocytes and then transfected into HEK293T cell line to confirm its expression.Results Primary astrocytes were successfully isolated and cultured in vitro with a purity of 95%or above.Foxp1 CDS in astrocytes was cloned and compared with the CDS of forth Foxp1 transcript,showed a consecutive bases AGC insert at 514/515.New cloned Foxp1 CDS was found expressed at about 106 kD when the expression vector containing cloned Foxp1 CDS region was transfected to HEK293T cell line.Conclusions Primary astrocyte is uccessfully isolated and cultured and a new CDS of Foxp1 is obtained.