摘要
目的 :为了将雄性胎鼠组织细胞移植进入雌性受体鼠 ,Y染色体特异的性别决定区蛋白基因 (sexde terminingregionprotein ,Sry)作为常用的遗传标志需要在细胞分离前快速检测 ;为了检测孕 14d胎鼠的Sry基因 ,我们建立了一种快速的PCR方法。基于C5 7BL/ 6J小鼠Sry基因序列 ,我们设计了特异检测引物 ,并优化了PCR的反应条件包括引物和循环参数等。方法和结果 :模板的制备时间约 30min。取约 1mg胚胎组织 ,以 10mmol/LTris10mmol/LEDTApH 8 0缓冲液洗涤 2次 ,然后以 2 0 0 μL裂解液 ( 2 0mg/LproteinaseK ,0 5 %NP - 4 0 ,and 0 0 5 %Tween 4 0 )悬浮 ,再在 6 0℃孵育 15min以消化蛋白 ,95℃以上 5min灭活蛋白酶K。取 5 - 10 μL作为模板。PCR反应总体积为 5 0μL ,使用两套引物同时扩增 ,一套扩增Sry目的基因片段 ,另一套扩增IL - 3基因片段作为内对照。扩增时间为 1 5h。扩增产物 10 - 15 μL采用 2 5 % - 3%的琼脂糖凝胶在 12 - 15V/cm的电压下电泳。 10min即可观察电泳结果。根据引物设计判断结果 ,Sry目的基因片段为 4 4 4bp、IL - 3基因片段为 6 4 9bp。操作约 10个胚胎总时间小于 3 5h。PCR扩增的特异性与特异探针荧光原位杂交结果一致。结论 :该PCR方法为检测胎鼠性别决定区蛋白基因的快速、简单?
AIM: To detect quickly the Y-chromosome specific sex determining region protein (Sry) gene in mouse fetuses on embryonic day 14.5 with a PCR method. METHODS: We designed specific primers with the OLIGO5.0 software. Templates were prepared in 30 minutes by the following way. About 1 mg embryonic tissue but not fetal liver was suspended, and treated with 200 μL of lysis buffer, consisting of PCR buffer containing 20 mg/L proteinase K, 0.5% NP-40, and 0.05% Tween 40, at 60℃ for 15 minutes, heated for 5 minutes at 100℃, 10 μL was used as template. The PCR reaction was performed in 50 μL, using two sets of primers specific for Sry gene (chromosome Y) and IL-3 gene (chromosome 11). PCR conditions and cycle numbers were optimized. The assessment of the results was done by electrophoresis in 3% agarose run at high voltage. The specificity of the method was confirmed by fluorescent in situ hybridization (FISH) using a specific male probe on embryonic tissue cells. RESULTS: Electrophoresis showed that PCR product of male control DNA consisted of a 649 bp product representing the IL-3 gene and a 444 bp product representing the Y-specific Sry gene, female control DNA only one 649 bp product. Fetuses with two bands matching those as seen in male control DNA are the presumptive male fetuses. Fetuses, only the IL-3-associated 649 bp band, are the presumptive female fetuses. These were confirmed by FISH. The entire procedure took <3.5 h. CONCLUSION: The established PCR assay offers a quick, simple, accurate, and sensitive detection of sex determining region protein gene in mouse fetuses. This method allowed the preparation and culture of pure male and female hematopoietic stem cells from fetal tissue.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2003年第5期622-626,T003,共6页
Chinese Journal of Pathophysiology
基金
ScienceandTechnologyFoundationofGuang dongprovince (No.99M0 12 0 4G)
