Ramírez Miguel A, Pericuesta Eva, Fernandez-Gonzalez Raul, Moreira Pedro, Pintado Belen, Gutierrez-Adan Alfonso
Departamento de Reproducción Animal, INIA, Ctra, De La Coruña Km 5,9, Madrid 28040, España.
Reprod Biol Endocrinol. 2006 Nov 8;4:55. doi: 10.1186/1477-7827-4-55.
In the mouse, culture of embryonic stem (ES) cells may decrease their pluripotency and give rise to foetal abnormalities in recipient embryos. These abnormalities are frequently associated with both, chromosome abnormalities or epigenetic alteration of imprinting genes; however, little is known about the epigenetic stability of endogenous retrotransposable elements (REs). In our laboratory, we came across a R1 ES cell line, which at passage 27, lost the ability of germline transmission and started inducing the kinky tail phenotype in all chimeric animals produced with it.
In order to investigate whether this phenotype was associated with chromosome alteration, inadvertent differentiation, or epigenetic modification, we characterized and compared this R1 ES cell line at passage 27 with an early passage and with a second ES cell line C57/CBAF1 generated in our laboratory. We assessed: i) karyotype; ii) expression of pluripotent and differentiation markers, iii) mRNA transcription by qRT-PCR of two REs, intracisternal-A particle (IAP) and murine endogenous-retrovirus-L (MuERV-L), and iv) methylation of IAP and MuERV-L.
The R1 ES cell at passage 27, presented normal morphology, karyotype, and expression of genetic markers characteristic of pluripotent; however, it was detected an altered mRNA transcription of sense and antisense RNA strands of both REs, concomitantly with an altered methylation pattern for the IAP element but not for MuERV-L. These results indicate that besides methylation, other post-transcriptional processes are involved in gene silencing of some REs; and that culture of ES cells may decrease their pluripotency by producing inadvertent alterations in the expression of REs without significantly affecting the morphology, chromosome structure, and expression of pluripotent or differentiation markers.
Inadvertent REs instability may have important consequences for the use of ES cells in transgenesis (chimera formation) or in cell therapy.
在小鼠中，胚胎干细胞（ES细胞）的培养可能会降低其多能性，并导致受体胚胎出现胎儿异常。这些异常通常与染色体异常或印记基因的表观遗传改变有关；然而，关于内源性逆转座子元件（REs）的表观遗传稳定性知之甚少。在我们实验室，我们遇到了一个R1 ES细胞系，在传代27次时，它失去了种系传递能力，并开始在所有与之产生的嵌合动物中诱导出扭尾表型。
为了研究这种表型是否与染色体改变、意外分化或表观遗传修饰有关，我们对传代27次的这个R1 ES细胞系与早期传代的细胞系以及我们实验室产生的第二个ES细胞系C57/CBAF1进行了表征和比较。我们评估了：i）核型；ii）多能性和分化标志物的表达，iii）通过qRT-PCR对两种REs，即脑内A颗粒（IAP）和小鼠内源性逆转录病毒-L（MuERV-L）进行mRNA转录分析，以及iv）IAP和MuERV-L的甲基化情况。
传代27次的R1 ES细胞呈现出正常的形态、核型以及多能性特征性遗传标志物的表达；然而，检测到两种REs的正义和反义RNA链的mRNA转录均发生改变，同时IAP元件的甲基化模式发生改变，但MuERV-L未改变。这些结果表明，除了甲基化之外，其他转录后过程也参与了某些REs的基因沉默；并且ES细胞的培养可能会通过在REs表达中产生意外改变而降低其多能性，而不会显著影响形态、染色体结构以及多能性或分化标志物的表达。
REs的意外不稳定性可能会对ES细胞在转基因（嵌合体形成）或细胞治疗中的应用产生重要影响。
