Iatrou K, Meidinger R G
Department of Medical Biochemistry, University of Calgary.
Proc Natl Acad Sci U S A. 1990 May;87(10):3650-4. doi: 10.1073/pnas.87.10.3650.
A pair of silkmoth chorion chromosomal genes, HcA.12-HcB.12, was inserted into a baculovirus transfer vector, pBmp2, derived from the nuclear polyhedrosis virus of Bombyx mori. This vector, which permits the insertion of foreign genetic material in the vicinity of a mutationally inactivated polyhedrin gene, was used to acquire the corresponding recombinant virus. Injection of mutant silkmoth pupae that lack all Hc chorion genes with the recombinant virus resulted in the infection of all internal organs including follicular tissue. Analysis of RNA from infected tissues has demonstrated that the two chorion genes present in the viral genome are correctly transcribed under the control of their own promoter in follicular cells, the tissue in which chorion genes are normally expressed. The chorion primary transcripts are also correctly processed in the infected follicular cells and yield mature mRNAs indistinguishable from authentic chorion mRNAs present in wild-type follicles. These results demonstrate that recombinant nuclear polyhedrosis viruses can be used as transducing vectors for introducing genetic material of host origin into the cells of the organism and that the transduced genes are transiently expressed in a tissue-specific manner under the control of their resident regulatory sequences. Thus we show the in vivo expression of cloned genes under cellular promoter control in an insect other than Drosophila melanogaster. The approach should be applicable to all insect systems that are subject to nuclear polyhedrosis virus infection.
将一对家蚕卵壳染色体基因HcA.12 - HcB.12插入到源自家蚕核型多角体病毒的杆状病毒转移载体pBmp2中。该载体允许在一个经突变失活的多角体蛋白基因附近插入外源遗传物质，用于获得相应的重组病毒。用重组病毒注射缺乏所有Hc卵壳基因的突变家蚕蛹，导致包括滤泡组织在内的所有内部器官被感染。对感染组织的RNA分析表明，病毒基因组中存在的两个卵壳基因在滤泡细胞（卵壳基因正常表达的组织）中，在其自身启动子的控制下被正确转录。卵壳初级转录本在受感染的滤泡细胞中也被正确加工，并产生与野生型滤泡中存在的真实卵壳mRNA难以区分的成熟mRNA。这些结果表明，重组核型多角体病毒可用作转导载体，将宿主来源的遗传物质引入生物体细胞，并且转导的基因在其自身调控序列的控制下以组织特异性方式瞬时表达。因此，我们展示了在除黑腹果蝇之外的昆虫中，克隆基因在细胞启动子控制下的体内表达。该方法应适用于所有遭受核型多角体病毒感染的昆虫系统。
