【摘要】 目的　为筛选、克隆小鼠短指 /缺指畸形肢体的特异表达基因 ,构建了GD18小鼠畸形肢体 (短指 /缺指 )与正常肢体的差异表达的cDNA消减文库。方法　应用抑制消减杂交技术 (SSH ) ,结合PCRcDNA合成法 ,将从总RNA中合成的双链cDNA ,酶切成平均大小为 4 0 0～ 6 0 0bp的片段 ,经接头连接 ,两次消减杂交和两次抑制性PCR后 ,将产物与T/A载体连接构建cDNA消减文库 ,并转染大肠杆菌进行文库扩增 ,菌液PCR筛选有插入片段的SSH克隆。结果　成功地构建了GD18小鼠畸形肢体(短指 /缺指 )差异表达的cDNA消减文库 ,从中挑取2 75个克隆进行菌液PCR分析 ,结果显示其中 2 5 5均得到 2 0 0～ 6 0 0bp插入片段。结论　SSH与PCR合成双链cDNA ,是一种自微量总RNA中构建高特异性的差异表达cDNA消减文库的有效方法。消减文库的构建 ,有助于筛选、克隆小鼠短指 /缺指畸形肢体的特异表达基因更多还原

【Abstract】 AIM For screening and cloning specific gene of mouse with brachydactyly/oligodactyly, a cDNA subtraction library of GD18 mouse embryo malformed limbs(brachydactyly/oligodactyly) was constructed. METHODS Total RNA were isolated from tissue of malformed and normal limbs respectively. Doublestrand cDNA were synthesized from total RNA using the PCR cDNA synthesis method. The dscDNA then were RsaⅠ digested and divided into two groups and ligated to the specified adaptor 1 and adaptor 2R respectively. After hybridized twice,underwent two times of suppression PCR,with arms of T/A plasmid vectors and transfection of E.coli DH5α to set up the subtractive library. RESULTS Forward and reverse subtractive library of GD18 mouse embryo with brachydactyly/oligodacty was constructed successfully. 275 white clones were picked randomly and 255 clones contain 200-600 bp inserts. CONCLUSION SSH combined with the PCR cDNA synthesis from total RNA is an efficient protocol to construct a highly specific cDNA subtractive library. The construction of the library of GD18 mouse embryo brachydactyly/oligodactyly limbs tissue lays the foundations for screening and cloning specific gene of mouse with brachydactyly/oligodactyly.更多还原