Targeted transgene integration into transgenic mouse fibroblasts carrying the full-length human AAVS1 locus mediated by HSV/AAV rep+ hybrid amplicon vector
Targeted transgene integration into transgenic mouse fibroblasts carrying the full-length human AAVS1 locus mediated by HSV/AAV rep+ hybrid amplicon vector
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DOI：
10.1038/sj.gt.3302061
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发表时间：
2003-09-01
期刊：
影响因子：
5.1
通讯作者：
Breakefield, XO
中科院分区：
文献类型：
--
作者：
Bakowska, JC;Di Maria, MV;Breakefield, XO
含有AAV反向末端重复序列（ITR）和rep基因序列的单纯疱疹病毒1型/腺相关病毒（HSV/AAV）rep（+）杂合扩增子载体可以介导位点特异性整合到人类基因组中。在这项研究中，我们已经产生和特点的第一个转基因小鼠，承担全长（8.2 kb）的人AAVS 1基因座。将来自该小鼠系的永生化小鼠胚胎成纤维细胞用rep（+）、rep（仅含有侧接转基因的ITR）杂交扩增子载体和标准扩增子载体转导，以确定稳定的整合频率和整合位点。转基因成纤维细胞的转导导致rep（+）杂合扩增子载体的稳定整合频率比rep或标准扩增子载体高10倍。来自用rep（+）杂合扩增子载体稳定转导的转基因细胞的基因组DNA的Southern印迹分析揭示了在50%的克隆中转基因在AAVS 1基因座处的位点特异性整合。一些位点特异性和随机整合事件限于ITR侧翼的转基因盒。相比之下，用rep或标准扩增子载体转导转基因小鼠细胞导致整个rep杂合扩增子或扩增子DNA的随机整合，所述扩增子DNA作为各种大小的串联体掺入宿主基因组中。这些结果第一次证明，携带人AAVS 1基因座的转基因小鼠的基因组充当在Rep.
Herpes simplex virus type 1/adeno-associated virus (HSV/AAV) rep(+) hybrid amplicon vectors containing AAV inverted terminal repeats (ITRs) and rep gene sequences can mediate site-specific integration into the human genome. In this study, we have generated and characterized the first transgenic mice that bear the full-length (8.2 kb) human AAVS1 locus. Immortalized mouse embryonic fibroblasts from this mouse line were transduced with the rep(+), rep (containing only ITRs flanking the transgene) hybrid amplicon vectors, and the standard amplicon vector to determine stable integration frequency and the site of integration. Transduction of transgenic fibroblasts resulted in a 10-fold higher stable integration frequency with rep(+) hybrid amplicon vector than with rep or standard amplicon vectors. Southern blot analysis of genomic DNA from transgenic cells stably transduced with the rep(+) hybrid amplicon vector revealed site-specific integration of transgenes at the AAVS1 locus in 50% of clones. Some site-specific and random integration events were limited to the ITR-flanked transgene cassette. In contrast, transduction of transgenic mouse cells with the rep or standard amplicon vectors resulted in random integrations of the entire rep hybrid amplicon or amplicon DNA that were incorporated into the host genome as a concatenate of various sizes. These results demonstrate for the first time that the genome of transgenic mice bearing the human AAVS1 locus serves as a platform for site-specific integration of AAV ITR-flanked transgene cassettes within the hybrid amplicon vector in the presence of Rep.