Knockdown of porcine endogenous retrovirus (PERV) expression by PERV-specific shRNA in transgenic pigs
Knockdown of porcine endogenous retrovirus (PERV) expression by PERV-specific shRNA in transgenic pigs
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DOI：
10.1111/j.1399-3089.2008.00442.x
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发表时间：
2008-02-01
影响因子：
3.9
通讯作者：
Denner, Joachim
中科院分区：
文献类型：
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作者：
Dieckhoff, Britta;Petersen, Bjoern;Denner, Joachim
工作背景：使用猪细胞、组织或器官的异种移植可能与猪内源性逆转录病毒（PERV）的传播有关。在猪基因组中已经鉴定出超过50个病毒拷贝，并且从猪细胞中释放出三种不同亚型的PERV，其中两种能够在体外感染人类细胞。RNA干扰是一个很有前途的选择，以抑制PERV transmission.Methods：我们最近选择了一个有效的si（小干扰）RNA对应于一个高度保守的区域在PERV DNA，这是能够抑制表达的所有PERV亚型在PERV感染的人类细胞，以及在原代猪细胞。使用表达相应sh（短发夹）RNA的慢病毒载体转染猪成纤维细胞，并通过体细胞核转移克隆产生转基因猪。结果：7头仔猪均整合了外源基因，并通过实时荧光定量RT-PCR检测了PERV和shRNA的表达。shRNA的表达被发现在所有的组织调查和PERV的表达被显着抑制时，与野生型control animals.Conclusion相比：这种策略可能会导致动物兼容PERV安全异种移植。
Background: Xenotransplantation using porcine cells, tissues or organs may be associated with the transmission of porcine endogenous retroviruses (PERVs). More than 50 viral copies have been identified in the pig genome and three different subtypes of PERV were released from pig cells, two of them were able to infect human cells in vitro. RNA interference is a promising option to inhibit PERV transmission.Methods: We recently selected an efficient si (small interfering) RNA corresponding to a highly conserved region in the PERV DNA, which is able to inhibit expression of all PERV subtypes in PERV-infected human cells as well as in primary pig cells. Pig fibroblasts were transfected using a lentiviral vector expressing a corresponding sh (short hairpin) RNA and transgenic pigs were produced by somatic nuclear transfer cloning. Integration of the vector was proven by PCR, expression of shRNA and PERV was studied by in-solution hybridization analysis and real-time RT PCR, respectively.Results: All seven born piglets had integrated the transgene. Expression of the shRNA was found in all tissues investigated and PERV expression was significantly inhibited when compared with wild-type control animals.Conclusion: This strategy may lead to animals compatible with PERV safe xenotransplantation.