SSH方法分离大剂量照射后小鼠骨髓差异表达EST序列

Expressed sequence tags isolated from mouse bone marrow cells irradiated with high-dose gamma rays by suppression subtractive hybridization (SSH)

目的 为探讨骨髓辐射损伤的分子机理 ,研究了整体照射条件下 ,辐射前后骨髓基因表达的变化。方法 小鼠 7Gyγ射线照射后 4h提取骨髓总RNA为实验方 (tester) ,对照组动物骨髓总RNA为驱动方 (driver) ;实验方、驱动方总RNA由SMARTcDNA合成方法合成双链cDNA ,进而由抑制消减杂交 (SSH)方法获得消减杂交产物 ,消减杂交产物克隆到T载体建立消减文库 ;对部分克隆进行杂交筛选及序列比对。结果 消减杂交文库挑取 80 0个克隆进行PCR扩增 ,阳性率约为86 % ;部分克隆经两轮杂交筛选获得 1 4个差异片段 ;7个差异片段与鼠EST库已有序列高度相似 ,其余 7个差异片段可能是新的EST序列。

Objective\ To explore the molecular mechanism of bone marrow injury caused by irradiation, gene expression of bone marrow cells in mice after whole body irradiation was studied. Methods\ Total RNA extracted from bone marrow cells in mice at 4 h after whole body irradiation with 7 Gy γ\|rays was taken as the tester and the total RNA from control mice as the driver cDNA subtraction was performed using the protocols described in the Clontech SMART PCR cDNA Sythesis Kit and PCR\|select cDNA Subtraction Kit.The subtracted cDNA was then inserted into T vector to generate subtracted cDNA library.Clones of the subtracted cDNA library were screened by hybridization and the insert sequence of the positive clones was compared with the sequence in the GenBank. Results\ A total of 800 clones selected from the subtracted cDNA library were PCR\|amplified and about 86% had inserts.Fourteen differential cDNA fragments were acquired after two round hybridization screening,7 of them showed high similarity to the expressed sequence tags (ESTs) in the mouse EST database and the other 7 cDNA fragments were possibly new ESTs. Conclusion\ The succesessfully constructed subtracted cDNA library of bone marrow cells in mice after whole body high\|dose irradiation and the validation of some differential ESTs establish the basis for further research of radiation\|related genes.\;