A model for the mechanism of precise integration of a microinjected transgene
A model for the mechanism of precise integration of a microinjected transgene
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DOI：
10.1007/bf01969706
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发表时间：
1996-05-01
影响因子：
3
通讯作者：
Wilson, JB
中科院分区：
文献类型：
--
作者：
Mcfarlane, M;Wilson, JB
一种独特的转基因小鼠品系以非常精确的方式进行了转基因整合。该品系小鼠显示的表型遵循已证实的X连锁转基因插入的预测遗传模式。为了研究整合的机制，已经确定了转基因和细胞连接的DNA序列。在插入位点处野生型和转基因突变体序列之间的比较揭示，在转基因整合后细胞DNA没有丢失或重排。转基因5'和3'连接处的细胞序列在野生型中是连续的。整合物以首尾串联二聚体形式存在，与注入的单体相比，序列损失最小。对插入位点的分析揭示了转基因的5'端与细胞序列之间的5bp同源性。此外，在细胞序列内的插入位点附近，有几个涉及重组事件的序列基序，包括I型DNA拓扑异构酶的强共有位点的聚类和与人小卫星共有核心序列同源的区域、大肠杆菌Chi位点和鼠主要组织相容性复合体的EP基因内的减数分裂重组热点。这种特征的聚集很可能是插入事件完整性的因素。提出了一种描述这种精确整合机制的模型。
A unique transgenic mouse line has undergone transgene integration in a very precise fashion. The phenotype displayed by mice of the line followed the predicted inheritance patterns for X-linked transgene insertion which has been confirmed. In order to investigate the mechanism of integration the DNA sequence of the transgene and cellular junctions have been determined. A comparison between wild type and transgenic mutant sequences at the site of insertion revealed that there was no loss or rearrangement of cellular DNA upon integration of the transgene. The cellular sequences at the transgene 5' and 3' joins are contiguous in the wild type. The integrant exists as a head to tail tandem dimer with minimal loss of sequence compared with the injected monomer. Analysis of the site of insertion has revealed a 5 bp homology between the 5' end of the transgene and the cellular sequences. In addition, adjacent to the site of insertion within the cellular sequences, there are several sequence motifs implicated in recombination events including a clustering of strong consensus sites for DNA topoisomerase type I and a region of homology to the human minisatellite consensus core sequence, the Escherichia coli Chi site and the meiotic recombination hotspot within the EP gene of the murine major histocompatibility complex. This clustering of features is likely to have been factorial in the integrity of the insertion event. A model depicting the mechanism of this precise integration is proposed.