Robert Koch-Institute, Berlin, Germany.
Xenotransplantation. 2012 Mar-Apr;19(2):112-21. doi: 10.1111/j.1399-3089.2012.00683.x.
Porcine endogenous retroviruses (PERVs) represent a risk of xenotransplantation using porcine cells, tissues, or organs, as they are integrated in the porcine genome and have been shown to be able to infect human cells in vitro. To increase viral safety by RNA interference, transgenic pigs expressing a PERV-specific small hairpin (sh)RNA targeted to a highly conserved sequence in the pol gene (pol2) were generated in which expression of PERVs was reduced (Xenotransplantation, 15, 2008, 38). However, it remains to be shown how long expression of the shRNA and the RNA interference is effective in reducing PERV expression.
To analyze the long-term duration of RNA interference, expression of the PERV-specific pol2 shRNA and inhibition of PERV expression was studied repeatedly in fibroblasts and peripheral blood mononuclear cells (PBMCs) of transgenic pigs over a period of 3 yr, when animals were sacrificed and expression was studied in different organs. Expression of the PERV-specific shRNA was measured using a newly developed real-time PCR, and expression of PERV was measured using a PERV-specific real-time PCR.
Over a period of 3 yr, PERV-specific shRNA and green fluorescent protein (GFP) as reporter of the vector system were consistently expressed in transgenic animals. PERV expression was significantly reduced during the entire period. Levels of PERV and shRNA expression were different in the various organs. PERV expression was highest in the spleen and the lungs and lowest in liver and heart. However, in all organs of the transgenic pigs, PERV expression was inhibited compared with the vector control animals.
Transgenic pigs expressing PERV-specific shRNA maintained their specific RNA interference long term, suggesting that PERV expression in the xenotransplants will be suppressed over extended periods of time.
猪内源性逆转录病毒（PERV）代表了使用猪细胞、组织或器官进行异种移植的风险，因为它们整合在猪基因组中，并已被证明能够在体外感染人类细胞。为了通过 RNA 干扰增加病毒安全性，生成了表达针对 pol 基因（pol2）中高度保守序列的 PERV 特异性短发夹（sh）RNA 的转基因猪，其中 PERV 的表达减少（《异种移植》，2008 年，38 期）。然而，表达 shRNA 和 RNA 干扰在降低 PERV 表达方面的有效性需要进一步证明。
为了分析 RNA 干扰的长期持续时间，在 3 年内，反复研究了转基因猪的成纤维细胞和外周血单核细胞（PBMC）中 PERV 特异性 pol2 shRNA 的表达和 PERV 表达的抑制情况，当动物被处死时，在不同的器官中研究了表达情况。使用新开发的实时 PCR 测量 PERV 特异性 shRNA 的表达，使用 PERV 特异性实时 PCR 测量 PERV 的表达。
在 3 年内，转基因动物中持续表达 PERV 特异性 shRNA 和绿色荧光蛋白（GFP）作为载体系统的报告基因。在整个期间，PERV 的表达显著降低。在不同的器官中，PERV 和 shRNA 的表达水平不同。PERV 的表达在脾脏和肺部最高，在肝脏和心脏最低。然而，与载体对照动物相比，转基因猪的所有器官中的 PERV 表达均受到抑制。
表达 PERV 特异性 shRNA 的转基因猪长期维持其特异性 RNA 干扰，表明异种移植中的 PERV 表达将在较长时间内受到抑制。
