Instability of the insertional mutation in CftrTgH(neoim)Hgu cystic fibrosis mouse model.
Instability of the insertional mutation in CftrTgH(neoim)Hgu cystic fibrosis mouse model.
复制标题
CftrTgH(neoim)Hgu 囊性纤维化小鼠模型中插入突变的不稳定性。
DOI：
10.1186/1471-2156-5-6
复制
发表时间：
2004-04-21
期刊：
影响因子：
2.9
通讯作者：
Tümmler, B
中科院分区：
文献类型：
--
作者：
Charizopoulou, N;Jansen, S;Dorsch, M;Stanke, F;Dorin, JR;Hedrich, HJ;Tümmler, B
利用胚胎干细胞中的基因靶向产生各种小鼠模型，极大地推动了囊性纤维化疾病的研究。此外，在产生同源菌株的不同近交菌株上引入相同的突变有助于寻找修饰基因。从原始的 CftrTgH(neoim)Hgu CF 小鼠模型中，我们使用严格的兄弟 × 姐妹交配两个近交 CftrTgH(neoim)Hgu 小鼠品系（CF/1 和 CF/3）生成。此后，插入突变从 CF/3 渐渗到三个近交背景（C57BL/6、BALB/c、DBA/2J）中，产生同源动物。在每个回交周期中，通过 Southern RFLP 直接探测插入来监测插入突变的种系传递。为了绕过这个耗时的程序，我们设计了一种基于 PCR 的替代方案，通过与破坏的基因座紧密连锁的 Cftr 基因内微卫星基因型，在 Cftr 基因座上区分小鼠品系。使用这种方法，我们能够根据三个近交系和 Cftr 基因座突变体 CftrTgH(neoim)Hgu 的差异单倍型背景来识别携带插入突变的动物。此外，该方法有助于在 57 只分型动物中的两只中鉴定出从破坏的 Cftr 基因座中精确切除的载体。这种向野生型状态的恢复没有任何序列丢失，揭示了同源动物生产过程中插入突变的不稳定性。我们提出基因内微卫星标记作为一种工具，用于在同源动物育种过程中快速有效地识别受体菌株中感兴趣的基因座。此外，相同的基因分型方法可以识别载体切除事件，从而对小鼠中插入突变的稳定性提出疑问。
A major boost to the cystic fibrosis disease research was given by the generation of various mouse models using gene targeting in embryonal stem cells. Moreover, the introduction of the same mutation on different inbred strains generating congenic strains facilitated the search for modifier genes. From the original CftrTgH(neoim)Hgu CF mouse model we have generated using strict brother × sister mating two inbred CftrTgH(neoim)Hgu mouse lines (CF/1 and CF/3). Thereafter, the insertional mutation was introgressed from CF/3 into three inbred backgrounds (C57BL/6, BALB/c, DBA/2J) generating congenic animals. In every backcross cycle germline transmission of the insertional mutation was monitored by direct probing the insertion via Southern RFLP. In order to bypass this time consuming procedure we devised an alternative PCR based protocol whereby mouse strains are differentiated at the Cftr locus by Cftr intragenic microsatellite genotypes that are tightly linked to the disrupted locus. Using this method we were able to identify animals carrying the insertional mutation based upon the differential haplotypic backgrounds of the three inbred strains and the mutant CftrTgH(neoim)Hgu at the Cftr locus. Moreover, this method facilitated the identification of the precise vector excision from the disrupted Cftr locus in two out of 57 typed animals. This reversion to wild type status took place without any loss of sequence revealing the instability of insertional mutations during the production of congenic animals. We present intragenic microsatellite markers as a tool for fast and efficient identification of the introgressed locus of interest in the recipient strain during congenic animal breeding. Moreover, the same genotyping method allowed the identification of a vector excision event, posing questions on the stability of insertional mutations in mice.
登录
查看更多内容
影响因子：
14.9
作者：
Corrette-Bennett, SE;Mohlman, NL;Lahue, RS
通讯作者：
Lahue, RS
影响因子：
11.4
作者：
Delaney, SJ;Alton, EWFW;Wainwright, BJ
通讯作者：
Wainwright, BJ
影响因子：
2.5
作者：
DORIN, JR;STEVENSON, BJ;PORTEOUS, DJ
通讯作者：
PORTEOUS, DJ
DOI：
10.1111/j.1432-1033.1973.tb02881.x
发表时间：
1973-01-01
期刊：
EUROPEAN JOURNAL OF BIOCHEMISTRY
影响因子：
--
作者：
GROSS-BELLARD, MM;OUDET, P;CHAMBON, P
通讯作者：
CHAMBON, P
影响因子：
30.8
作者：
Rozmahel, R;Wilschanski, M;Tsui, LC
通讯作者：
Tsui, LC