GJB2基因条件敲除小鼠的繁殖、基因型鉴定及听力检测  被引量:3

Breeding,Genotyping and Hearing Examination of the GJB2 Conditional Knock Out Mice

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作  者:万亚蕊[1,2] 张延平[2] 张晓强[1] 杨仕明[1] 

机构地区:[1]解放军总医院耳鼻咽喉头颈外科,北京100853 [2]解放军第309医院耳鼻喉科

出  处:《听力学及言语疾病杂志》2013年第5期505-508,共4页Journal of Audiology and Speech Pathology

基  金:全军十二五面上项目(11MS013);国家自然科学基金面上项目青年基金(30700936);国家自然科学基金重点项目(30730040)联合资助;中国人民解放军第309医院院内基金(LCB08MS12;309-09-03);国家973计划重大科学研究计划干细胞项目(2012CB967900);国家973计划重大科学问题导向项目(2011CBA01000);国家863计划专题项目(2007AA02Z150)

摘  要:目的探讨GJB2基因条件敲除(conditional connexin 26knock out,cCx26KO)小鼠的饲养、繁殖及基因型鉴定方法,为进一步研究GJB2基因突变导致非综合征型聋的机制奠定基础。方法将引进的两对转基因小鼠Cx26loxp/loxp和Pax2-Cre/+进行交配饲养与繁殖,选取子一代雌性Cx26loxp/-_Pax2-Cre/+小鼠与雄性小鼠Cx26loxp/loxp合笼交配,即获得cCx26KO小鼠。提取鼠尾组织基因组DNA,PCR方法鉴定动物基因型。采用c-ABR检测成年cCx26KO小鼠和野生型小鼠的听力,进一步验证PCR方法的正确性。结果 cCx26KO小鼠繁殖成功后,采用PCR方法鉴定分析,成功获得Cx26loxp/loxp_Pax2Cre/+、Cx26loxp/-_Pax2-Cre/+、Cx26loxp/loxp、Cx26loxp/-4种基因型小鼠,其繁殖结果符合孟德尔遗传定律。与野生型小鼠相比,cCx26KO小鼠c-ABR反应阈显著升高,约达95dB SPL。结论 PCR方法可准确鉴定子鼠的基因型,雌性Cx26loxp/-_Pax2-Cre/+小鼠与雄性Cx26loxp/loxp小鼠交配是获得cCx26KO实验小鼠的有效途径。Objective To explore the methods of breeding, reproduction and genotype of GJB2 knock--out (cCx26KO) mice and further study the critical role of G.IB2 mutation in the onset of nonsyndromic hearing loss (NSHL). Methods Two pairs of transgenie mice (Cx261oxp/loxp and Pax2--Cre/-+-) were inbreeded to produce Cx261oxp/--_Pax2--Cre/-? ones, female of which were used to mate with the male Cx261oxp/loxp ones to finally get the Cx261oxp/loxp_Pax2 -- Cre/q- mice (cCx26KO). The genotype was done by PCR and Agarose gel electro- phoresis using genome DNA extracted from the mice tails. The c--ABR was used to detect the hearing ability o[ the cCx26KO mice. Results Both breeding and reproduction of eCx26KO mice were successful. It was fruitful to obtain four genotype mice(Cx261oxp/loxp_ Pax2--Cre / q-, Cx261oxp / --_Pax2--Cre / -k, Cx261oxp/loxp, Cx261oxp / --) by the breeding Cx261oxp / -- Pax2Cre / -I- and Cx261oxp/loxp mice. The results of breeding were met with the Mendel's law. The c-- ABR revealed elevated response threshold around 95 dB SPL in cCx26KO mouse compared to the wild type ones, which further validated the accuracy of the PCR method. Conclusion The PCR method is cor- rectly identified sub pups genotype and the female Cx261oxp/--_Pax2--Cre/q- mice mating with the male Cx261oxp/ loxp ones is an effective way to obtain the cCx26KO mice.

关 键 词:GJB2基因 小鼠 基因敲除 野生型 基因型鉴定 感音神经性聋 

分 类 号:R373.21[医药卫生—病原生物学]

 

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