Sauvain Marc-Olivier, Dorr Alexander P, Stevenson Brian, Quazzola Alexandra, Naef Félix, Wiznerowicz Maciej, Schütz Frédéric, Jongeneel Victor, Duboule Denis, Spitz François, Trono Didier
Ecole Polytechnique Fédérale de Lausanne, School of Life Sciences, Lausanne CH-1015, Switzerland.
J Virol. 2008 Jul;82(14):7111-9. doi: 10.1128/JVI.00623-08. Epub 2008 May 7.
Lentivector-mediated transgenesis is increasingly used, whether for basic studies as an alternative to pronuclear injection of naked DNA or to test candidate gene therapy vectors. In an effort to characterize the genetic features of this approach, we first measured the frequency of germ line transmission of individual proviruses established by infection of fertilized mouse oocytes. Seventy integrants from 11 founder (G0) mice were passed to 111 first generation (G1) pups, for a total of 255 events corresponding to an average rate of transmission of 44%. This implies that integration had most often occurred at the one- or two-cell stage and that the degree of genotypic mosaicism in G0 mice obtained through this approach is generally minimal. Transmission analysis of eight individual proviruses in 13 G2 mice obtained by a G0-G1 cross revealed only 8% of proviral homozygosity, significantly below the 25% expected from purely Mendelian transmission, suggesting counter-selection due to interference with the functions of targeted loci. Mapping of 239 proviral integration sites in 49 founder animals revealed that about 60% resided within annotated genes, with a marked tendency for clustering in the middle of the transcribed region, and that integration was not influenced by the transcriptional orientation. Transcript levels of a set of arbitrarily chosen target genes were significantly higher in two-cell embryos than in embryonic stem cells or adult somatic cells, suggesting that, as previously noted in other settings, lentiviral vectors integrate preferentially into regions of the genome that are transcriptionally active or poised for activation.
慢病毒载体介导的转基因技术正越来越多地被使用，无论是用于基础研究，作为向受精卵原核注射裸DNA的替代方法，还是用于测试候选基因治疗载体。为了描述这种方法的遗传特征，我们首先测量了通过感染受精小鼠卵母细胞建立的单个原病毒的种系传递频率。来自11只奠基者（G0）小鼠的70个整合体被传递给111只第一代（G1）幼崽，总共发生了255次事件，平均传递率为44%。这意味着整合最常发生在单细胞或双细胞阶段，并且通过这种方法获得的G0小鼠的基因型嵌合程度通常很小。对通过G0-G1杂交获得的13只G2小鼠中8个单个原病毒的传递分析显示，只有8%的原病毒纯合性，显著低于纯孟德尔遗传预期的25%，这表明由于对靶向基因座功能的干扰而存在反选择。对49只奠基动物中239个原病毒整合位点的定位显示，约60%位于注释基因内，在转录区域中间有明显的聚集趋势，并且整合不受转录方向的影响。一组任意选择的靶基因的转录水平在双细胞胚胎中明显高于胚胎干细胞或成体体细胞，这表明，如先前在其他情况下所指出的，慢病毒载体优先整合到基因组中具有转录活性或易于激活的区域。
