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

小鼠和大鼠动物实验中的一个大问题是小鼠肝炎病毒（MHV）和仙台病毒等高传播和致命病毒的流行感染。目前，大多数动物实验都是用无特定病原体（SPF）的动物进行的，这些动物特别容易被这些病毒感染。克服这些问题的方法之一是建立对病毒感染具有遗传抗性的动物。因此，我们已经开始通过基因转移将易感的MHV小鼠转化为抗性小鼠。反义RNA被认为是通过与义RNA杂交来特异性抑制mRNA（义RNA）的翻译。在病毒复制过程中，反义RNA也可以通过阻止病毒复制过程中不可避免的基因的表达来抑制病毒复制。首先，我们试图通过在培养细胞中表达反义RNA，使培养细胞对MHV具有抗性。我们制备了54个与部分MHV RNA聚合酶基因相同的寡核苷酸及其互补的寡核苷酸，对这些寡核苷酸进行了纯化，并对该序列进行了20 ~ 36次串联重复。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，检测其反义RNA水平和对MHV感染的抗性。总的来说，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与抗MHV感染的关系，最终建立利用反义RNA使易感动物产生耐药的系统。

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.