ADAM10条件性基因打靶载体的构建和鉴定.pdf.pdf

厦门大学硕士学位论文 ADAMl0条件性基因打靶载体的构建和鉴定 ADAMIO条件性基因打靶载体的构建和鉴定研究生:林熠华导师: 王义权教授周常文教授【摘要】【目的】构建ADAMl0条件性基因打靶载体,为制备ADAMl0条件性基因敲除的小鼠模型和研究ADAMl0基因的功能奠定基础。【方法】运用长片段PCR技术,从129/SVJ小鼠基因组中分步扩增ADAMl0 基因第--#F显子及其两端作为同源臂的侧翼序列,。经测序鉴定后,将外显子以及上下游同源臂片段分别连接于基因打靶骨架载体 pL451上,构建了基因打靶中间载体pEFUD。再从pSSC9载体上酶切下HSV—TK 基因,连接于pEFUD,最终构建成ADAMl0条件性基因打靶载体pEFUD-TK。为了验证载体上正负双向选择基因的功能,分别以环状和线性pEFUD-TK为实验组,pSSC9、pL451和空转为对照组,采用脂质体2000(LP2000)转染人神经母细胞瘤细胞(SH_SY5Y),研究Neo基因和HSV-TK基因的表达活性。【结果】:克隆的ADAMl0基因第二外显子及侧翼序列的酶切图谱与公布的129/svJ小鼠相应序列的酶切图谱相一致。测定的序列与数据库公布的小鼠相应序列基本相同(%)。其中,第--#b显子序列与公布的小鼠序列完全一致。外显子与上、下游同源臂序列均正确地连接于pL451载体,来源于pSSC9的 HSV—TK基因经过测序均与报道的HSV-TK基因序列完全相同,因此成功构建了 ADAMl0条件性基因打靶载体。用LP2000转染SH-SY5Y细胞,G418(850ng/m1) 筛选lO天后,空转对照组细胞全部死亡,pEFUD-TK环状实验组、pEFUD-TK线性化实验组、pL451和pSSC9对照组均有少量细胞存活,并形成小的细胞集落; 细胞再经GCV(10ng/m1)筛选,72小时后,环状和线性pEFUD—TK实验组以及 pSSC9对照组均有大量细胞死亡,G418筛选后形成的细胞集落大部分消失, pL451对照组基本无细胞死亡,原有的小细胞集落继续生长。【结论】:成功构建并鉴定了ADAMl0基因条件性基因敲除打靶载体(pEFUD—TK),载体中正负双向选择基因能够发挥正常的功能。【关键词】条件性基因打靶载体构建ADAMl0基因 Construction and characterization of ADAMl0 conditional gene targeting vector Postgraduate:Lin zhaohua Supervisor:Wang yiquan Zhou Changwen Abs tract [Object]:Construction of ADAMIO conditional gene targeting vector is to lay the foundation of preparing the ADAMIO conditional knockout mice and to study the function of ADAMIO gene. [Material andMethod]:Genomic DNA fragments surrounding exon 2ofADAMIO gene were amplified from 129/SVJ mice by fragments ofwhich were short arm、 long arm and the exon2 were ligated tothe targeting skeleton vector pL451,respectively,to form the intermediate vector final target ing vector pEFUD-TK was constructed by ligating the HSV——TK gene fragment isolated from the pSSC9pl asmid to the vector pEFUD. In order to understand whether the Neo and HSV—。TKgene i nthe final construct play anormal role in vitor,the plasmid pEFUD—TK、pL451 and pSSC9 were separately transfected into human neuroblastoma cell s (SH—SY5Y)by LiPide2000(control gr