Spencer Shawal, Gugliotta Agustina, Koenitzer Jennifer, Hauser Hansjörg, Wirth Dagmar
Model Systems for Infection and Immunity, Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany.
Boehringer Ingelheim Pharma GmbH & Co. KG, Dep. Process Science Germany, Birkendorfer Str. 65, 88397 Biberach/Riß, Germany.
J Biotechnol. 2015 Feb 10;195:15-29. doi: 10.1016/j.jbiotec.2014.12.009. Epub 2014 Dec 20.
Intraclonal heterogeneity of genetically modified mammalian cells has been observed as a phenomenon that has a strong impact on overall transgene expression levels and that limits the predictability of transgene expression in genetically modified cells, thereby hampering single cell based screening approaches. The underlying mechanism(s) leading to this variance are poorly understood. To study the dynamics and mechanisms of heterogeneity of early stage silencing we analyzed the expression in more than 100 independent clones of CHOK1 cells that harbour genetically stable integrates of single copy reporter cassettes driven by EF1α and CMV promoters. Single cell analysis showed intraclonal variability with heterogeneity in expression in genetically uniform populations. DNA methylation is a well known mechanism responsible for silencing of gene expression. Interestingly, loss of expression was not associated with DNA methylation of the CMV promoter. However, in most of the clonal populations expression could be increased by inhibitors of the histone deacetylases (HDACi) suggesting that heterogeneity of transgene expression is crucially governed by histone modifications. Further, to determine if the epigenetic status of transgene expression is governed by the chromosomal integration locus we targeted heterologous expression cassettes into two chromosomal sites using recombinase mediated cassette exchange (RMCE). The expression status of a particular clone was faithfully re-established when the same promoter used. In this way the problem of early stage cell clone instability can be bypassed. However, upon introduction of an unrelated promoter methylation-independent silencing was observed. Together, these results suggest that histone modifications are the relevant mechanisms by which epigenetic modulation of transgene expression cassettes is governed in the early phase of clone generation.
已观察到基因改造的哺乳动物细胞存在克隆内异质性现象，该现象对整体转基因表达水平有强烈影响，并限制了转基因在基因改造细胞中表达的可预测性，从而阻碍了基于单细胞的筛选方法。导致这种差异的潜在机制尚不清楚。为了研究早期沉默异质性的动态变化和机制，我们分析了100多个独立的CHOK1细胞克隆中的表达情况，这些细胞克隆含有由EF1α和CMV启动子驱动的单拷贝报告基因盒的基因稳定整合体。单细胞分析显示，在基因均一的群体中存在克隆内变异性和表达异质性。DNA甲基化是一种众所周知的导致基因表达沉默的机制。有趣的是，表达缺失与CMV启动子的DNA甲基化无关。然而，在大多数克隆群体中，组蛋白脱乙酰酶抑制剂（HDACi）可提高表达，这表明转基因表达的异质性关键受组蛋白修饰调控。此外，为了确定转基因表达的表观遗传状态是否受染色体整合位点的控制，我们使用重组酶介导的盒式交换（RMCE）将异源表达盒靶向到两个染色体位点。当使用相同启动子时，特定克隆的表达状态能如实地重新建立。通过这种方式，可以绕过早期细胞克隆不稳定的问题。然而，引入无关启动子后，观察到与甲基化无关的沉默现象。总之，这些结果表明，组蛋白修饰是在克隆产生早期阶段调控转基因表达盒表观遗传调控的相关机制。
