A trial to convert the mouse susceptible to mouse hepahtis virus to resistant one by anti-sense RNA

在用小鼠和大鼠进行的动物实验中，一个很大的问题是具有高度传播性和致命性病毒的流行性感染，如小鼠肝炎病毒(MHV)和仙台病毒。目前，大多数动物实验都是使用无特定病原体(SPF)的动物进行的，这些动物特别容易感染这种病毒。克服这些问题的方法之一是建立对病毒感染具有遗传抵抗力的动物。因此，我们致力于通过基因转移将敏感小鼠转变为MHV耐药小鼠。反义RNA被认为是通过与正义RNA杂交来特异性地抑制mRNA(正义RNA)的翻译。在病毒复制中，反义RNA可能通过阻止病毒复制所必需的基因的表达来抑制病毒复制。首先，我们试图通过在MHV敏感的培养细胞中表达反义RNA来使其对MHV产生抗性。我们已经制备了与MHV RNA聚合酶基因部分相同的54mer寡核苷酸和与之互补的寡核苷酸，并将其命名为20-36个串联重复序列。将这些串联重复序列插入含有SV40或金属硫蛋白启动子的表达载体中，表达反义RNA，并与pSV2neo共转染L细胞。克隆在G418存在下生长的L细胞，并检测反义RNA水平和对MHV感染的抵抗力。总体而言，目前还没有观察到反义RNA水平与MHV抗性之间的相关性。我们希望进一步研究反义RNA与MHV感染抗性的关系，最终建立使易感动物对反义RNA产生抗性的体系。

A big problem in the animal experiments using mice and rats is epidemic infections with highly disseminating and fatal viruses as mouse hepatitis virus (MHV) and sendai virus. Nowadays, most of aminal experiments are carried out by using specific-pathogen free (SPF)animals which are especially prone to be infected with such viruses. One of the approaches to overcome such problems is to establish the animals which are genetically resistant to virus infections. Thus, we have undertaken to convert susceptible mice to MHV to resistant ones by gene transfer. Anti-sense RNA is considered to spceifically inhibit the translation of mRNA (sense RNA) by hybridizing to sense RNA. In the virus replication as well, antisense RNA may inhibit virus replication by preventing the expression of a gene which is inevitable to virus replication. First of all, we tried to make susceptible cultured cells to MHV resistant by expressing antisense RNA in such cultured cells. We have prepared 54mer oligonucleotides identical to a part of MHV RNA polymerase gene and oligonucleotides complementary to them, anealed these oligouncleotides and make 20 to 36 tandem repeats of such sequence. These tandem repeats were inserted into experssion vectors with SV40 or metallothionein promoters to express anti-sense RNA and they were cotransfected on L cells with pSV2 neo. L cells grown in the presence of G418 were cloned and examined for the level of anti-sense RNA and resistance to MHV infection. As a whole, no correlation has bee observed between the anti-sense RNA level and resistance to MHV at present. We would like to further investigate on the relationship between anti-sense RNA and resistance to MHV infection to finally establish the system to make susceptible animals to resistant by anti-sense RNA.