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罗金刚,,,,,,罗晓红,肖颂华,等.APPswe/PS1转基因模型鼠的基因分型与筛选研究[J].同济大学学报（医学版）,2012,33(5):22-27. [点击复制]
LUO Jin-gang,LUO Xiao-hong,XIAO Song-hua,et al.Genotyping and screening of APPswe/PS1 transgenic mouse[J].同济大学学报（医学版）,2012,33(5):22-27. [点击复制]

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APPswe/PS1转基因模型鼠的基因分型与筛选研究
罗金刚¹,,,,,,罗晓红²,肖颂华³,王英²,许英²,段朝晖²
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(1. 中山大学孙逸仙纪念医院检验科,广东广州 510120;南京军区福州总医院检验科,福建福州 350025;2. 中山大学孙逸仙纪念医院检验科,广东广州,510120;3. 中山大学孙逸仙纪念医院神经科,广东广州,510120)

摘要:

目的探讨老年性痴呆(Alzheimer disease,AD)转基因模型鼠Tg(APPswe PSEN1dE9)85Dbo的优化繁育和基因分型方法,筛选出适合AD研究的稳定转基因动物。方法 3种不同的繁殖配伍分组(组1,+/+×-/-♀;组2,+/+♀×-/-;组3,+/+♀×+/+)研究子代鼠的存活率及PS1转基因阳性率;提取子二代鼠尾基因组DNA并鉴定其质量,用双重PCR方法扩增PS1转基因片段,琼脂糖凝胶电泳观察结果;PCR产物测序证实其基因序列。结果改良后的方法全程耗时不到8 h,提取的基因组DNA在23 kb以上,完整性好,浓度为65～175 ng/μl。共繁殖子二代小鼠27窝计222只,存活206只,配伍组1子代存活率94.29%(99/105);配伍组2子代存活率91.67%(66/72);配伍组3子代存活率91.11%(41/45),3组间存活率差异无统计学意义(P=0.423>0.05)。3种配伍产生的子代鼠转基因阳性率都基本符合孟德尔遗传规律,转基因阳性率差异有统计学意义(P=0.028<0.05)。配伍组1子代鼠雄鼠阳性率65.1%(28/43),雌鼠阳性率35.7%(20/56),差异有统计学意义(P=0.003<0.05),性别与转基因阳性率相关(CO=0.280,P=0.004<0.05);配伍组2子代鼠,雄鼠阳性率56.3%(18/32),雌鼠阳性率55.9%(19/34),差异无统计学意义(P=0.586>0.05);配伍组3子代鼠,雄鼠阳性率81%(17/21),雌性阳性率65%(13/20),差异无统计学意义(P=0.212>0.05)。双重PCR产物经测序证实和预期完全一致。结论 Tg(APPswe,PSEN1dE9)85Dbo杂合子亲代配伍能产生存活至成年的纯合子子代鼠;改进后的基因分型可以作为小鼠基因分型的可靠方法,用于AD研究相关的胎鼠神经元培养实验,该动物模型杂合子雄鼠与野生型雌性鼠的配伍繁殖为最佳繁育筛选组合。

关键词: 阿尔茨海默病转基因小鼠基因分型双重PCR 杂合子

DOI：10.3969/j.issn1008 -0392.2012.05.005

投稿时间：2011-11-28

基金项目:广东省自然科学基金（8151008901000035);广东省科技计划（2010A030400006)

Genotyping and screening of APPswe/PS1 transgenic mouse

LUO Jin-gang¹,LUO Xiao-hong²,XIAO Song-hua³,WANG Ying²,XU Ying²,DUAN Chao-hui²

(1.Dept.of Clinical Laboratory,Sun Yat-sen Memorial Hospital,Sun Yat-sen University,Guangzhou 510120, Guangdong Province,China;2.Dept.of Clinical Laboratory,Fuzhou General Hospital of Nanjing Military Command, Fuzhou 350025,Fujian Province,China;3.Dept.of Clinical Neurology,Sun Yat-sen Memorial Hospital, Sun Yat-sen University,Guangzhou 510120,Guangdong Province,China)

Abstract:

Objective To optimize breeding and genotyping methods of Tg (APPswe，PSEN1dE9) 85Dbfor further study of Alzheimer disease (AD) . Methods FI generation mice were divided into 3 mating groups (group 1, + / + d X -/-$ \ group 2, + / + $ X -1-6) group 3, +/+$X+/+d). The genome DNA of F2 generation mice was isolated and genotyping was carried out by duplex PCR and agarose gel electrophoresis. The products of duplex PCR were sequenced. Results High quality genome DNA was obtained in less than 8 h for genotyping. 27 litters of F2 generation mice were breed with a total number of 222，206 of which survived to adulthood; the survival rates of group 1, 2 and 3 were 94. 29% (99/105)， 91.67% (66/72) and 91. 11% (41/45), respectively (P =0.423) . The transgenic positive results of three mating groups were accorded with Mendelian segregation. There were signivicant differences in transgenic positive rate among 3 mating groups (P =0. 028) . The transgenic positive rate in group 1 ( + / + 6 X -/-? ) was correlated with genders (CO =0. 280, P =0. 003, P <0. 05) , but not in other two groups. Products of duplex PCR verified by nucleotide sequencing were consistent as predicted. Conclusion Transgenic mating system ( + / + hemizygote X + / + hemizygote) brings out homozygotic progeny that survive till adults. The modified methods for DNA isolating and genotyping is reliable and time-saving. The best mating system for neuron culture of Tg (APPswe, PSEN1dE9) 85Dbo is + / + Hemizygote X -/ -sibling (male X female).

Key words: Alzheimer disease transgenic mice genotyping duplex PCR hemizygote