近交系基因敲除小鼠微卫星不稳定性分析

Analysis of microsatellite instability in inbred knockout mice

前期研究中选取了198个与基因突变和疾病相关的小鼠微卫星不稳定性位点,经对转基因和基因突变小鼠研究,发现有41个位点具有不稳定性。为了进一步确认这些位点与基因改变的相关性,本试验应用这41个微卫星位点对29种C57BL/6J基因敲除小鼠进行了检测,并将野生型C57BL/6J和129品系小鼠(ES供体)作为对照。通过PCR扩增,STR扫描和直接测序的方法检测微卫星的不稳定性。在41个微卫星位点中有10个位点在11种基因敲除小鼠中呈现不稳定性(24.4%,10/41)。核心序列为三核苷酸的D3Mit22在9号基因敲除小鼠中显示不稳定性,其余40个位点的核心序列均为二核苷酸,有9个位点呈现不稳定性(22.5%,9/40);另一方面,29种基因敲除小鼠有11种出现了不稳定性(37.9%,11/29);(TG)n为核心序列的二核苷酸表现不稳定性的比率最大(50%,2/4),依次为(GT)n(27.27%,3/11)和(AG)n(25%,1/4),重复序列(CA)n、(CT)n分别为23.08%(3/13)和20%(1/5)。克隆测序的结果显示,6种基因敲除小鼠呈现核心序列片段的插入,1种基因敲除小鼠则为缺失。有2个位点(D13Mit3和D14Mit102)在2种基因敲除小鼠中出现不稳定性,9号基因敲除小鼠在2个位点(D3Mit22和D13Mit3)呈现不稳定性。结果显示基因敲除小鼠出现了微卫星不稳定性。

Previously, we found that the genotypes are associated with mutation and disease, showed modified inbred mouse strain. To further confirm of 41 out of 198 mouse microsatellite loci,which microsatellite instability （MSI） in a genetically the relation between these loci and genetic chan ges,the 41 microsatellite loci were analyzed in 29 gene knockout （KO） mouse strains,wildtype C57BL/6J and 129 mouse strains （ES donor） served as controls. The MSI was detected by poly merase chain reaction （PCR）,short tandem sequence repeat （STR） scanning and direct sequence cloning. The results indicated that out of 41/10（24.4% ,10/41） loci showed MSI in 11 KO mouse strains. Except for the trinucleotide repeat locus of D3Mit22, which had microsatellite MSI in strain number 9,the core sequences of the remaining 40 loci were dinucleotide repeats,and out of 40 （22.5%,9/40）,9 showed MSI. out of 29（37.9%,11/29）KO mice strains Eleven, were recog nized as having MSI. The popular dinucleotide motifs in MSI were （TG）n（50M ,2/4） ,followed by （GT） （27. 27 %, 3/11 ） and （AG） n （ 25%, 1/4 ）. The microsatellite CMP in （ICA）. repeat was 23.08% （3/13） and （CT）, repeats were 20% （1/5）. According to cloning sequencing results, 6 KO mouse strains showed insertions of nucleotides whereas 1 showed a missing. Furthermore, 2 loci （D13Mit3 and D14Mit102） revealed MSI in 2 strains,and mouse strain number 9 showed MSI in two loci （D3Mit22 and D13Mit3） simultaneously. Collectively,these results indicated that mic rosatellite instability were present in the examined inbred KO mice.