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作者总结了51例AML和MDS中的FLT3基因突变情况,并分析了FLT3基因在Cos7细胞中转染后的表达和磷酸化情况。 材料和方法①白血病细胞和FLT3/ITD检测 从白血病细胞中抽提高分子量DNA,用PCR扩增复盖JM区的DNA片段,琼脂糖凝胶电泳上切下异常大小带并纯化,然后克隆进pMOS-Blue T-载体。选择10个重组子集落并在LB培养基中培养,而后制备质粒DNA,二条链借结合荧光素21 M13和T7引物在DNA测序仪上测序。用RT-PCR法检测FLT3转录本表达。异常大小产物同上克隆和测序,以确定转录本是在读框内。②转染子分析 借RT-PCR法扩增白血病细胞中复盖有突变FLT3,cDNA串联重复的Munl-EcoRV片段,扩增产物用Munl
The authors summarized the mutations of FLT3 gene in 51 cases of AML and MDS, and analyzed the expression and phosphorylation of FLT3 gene in Cos7 cells. Materials and Methods 1 Leukemia Cells and FLT3/ITD Assay Raised molecular weight DNA from leukemia cells, PCR amplified DNA fragments covering the JM region, anomalous size bands were cut on agarose gel electrophoresis and purified, then cloned into pMOS -Blue T-carrier. Ten recombinant colonies were selected and cultured in LB medium before plasmid DNA was prepared and the two strands were sequenced on a DNA sequencer by binding fluorescein 21 M13 and T7 primers. The expression of FLT3 transcripts was detected by RT-PCR. The abnormal size product was cloned and sequenced as above to confirm that the transcript was in reading frame. 2 Analysis of transfectants The RT-PCR method was used to amplify the MunL-Eco RV fragment covered with mutant FLT3 in the leukemic cells, and the amplified product was Munl