Tissue-specific expression of silkmoth chorion genes in vivo using Bombyx mori nuclear polyhedrosis virus as a transducing vector.

Abstract

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 moti. 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. Iijection 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 Drosophia melanogaster. The approach should be applicable to all insect systems that are subject to nuclear polyhedrosis virus infection.Genetic transformation represents a powerful methodological tool that facilitates the analysis of models of gene regulation. Although significant progress has been achieved in a number of plant and animal models for which methodology for stable or transient in vivo expression of cloned genes has been developed, confirmation of regulatory models of insect gene function has been hindered due to a lack of such expression systems. Thus far, only three types of in vivo expression systems have been developed for insect species. Two of them result in stable genetic transformation ofDrosophila melanogaster embryos and rely on the utilization of two transposable elements, P and hobo (1, 2). Because of their specificity, however, these systems are not applicable to other insect species. The third system, termed somatic transformation (3), relies on the direct injection of cloned gene sequences into Drosophila embryos and results in a transient expression of the injected genes in somatic cells. No comparable expression assays are available for any other insect systems.

Transgenesis approaches for functional analysis of peptidergic cells in the silkworm Bombyx mori

“…The first successful gene introduction in B. mori was accomplished by the recombinant BmSNPV-mediated in vivo expression of chorion genes controlled by their own regulatory elements. Infection of the silkworm pupae by the recombinant virus caused transient and tissue specific expression of chorion (Iatrou and Meidinger, 1990). …”

Analysis of developmentally regulated chorion gene promoter architecture via electroporation of silk moth follicles

“…To this end, we constructed several mutated A/B.L9 promoter regions and cloned them upstream of a reporter gene in order to introduce these plasmid constructs into silk moth follicle epithelial cells. The methods that have been used for transient expression of cloned moth genes are heterologous transformation in Drosophila using P‐element vectors (Mitsialis & Kafatos, ), transduction using nuclear polyhedrosis virus vectors (Iatrou & Meidinger, ), microinjection of plasmid‐carried genes in Bombyx embryos (Nikolaev et al ., ), the application of biolistic for the transformation of embryos and gonads (Thomas et al ., ), silk glands (Horard et al ., ), silkmoth follicle epithelial cells (Kravariti et al ., ) or larval and pupal tissues (Thomas et al ., ), and electroporation in B. mori embryonic tissues and in larval tissues such as ovaries or epithelial cells of larval imaginal wing discs (Thomas, ). In the present paper, we used and optimized the electroporation technique as an efficient and quick method for transient expression of plasmid constructs in Bombyx mori follicular epithelial cells instead of the biolistic method (Kravariti et al ., ).…”

Οργάνωση και ρύθμιση της έκφρασης των πρώιμων γονιδίων του χορίου του μεταξοσκώληκα Bombyx mori

“…Εντούτοις, η συντηρητικότητα των ρυθµιστικών µηχανισµών της ωογένεσης µεταξύ λεπιδοπτέρων και διπτέρων έχει επιτρέψει τη µελέτη των ρυθµιστικών περιοχών ορισµένων γονιδίων του χορίου του µεταξοσκώληκα στη Drosophila, µέσω ετερόλογου µετασχηµατισµού µε στοιχεία P (Mitsialis and Kafatos 1985;Mitsialis et al 1987Mitsialis et al , 1989Spoerel et al 1993). Εναλλακτικά, µια άλλη µέθοδος που έχει χρησιµοποιηθεί για το σκοπό αυτό, είναι η εισαγωγή και προσωρινή έκφραση κλωνοποιηµένων γονιδίων του χορίου στο µεταξοσκώληκα µέσω ραβδοϊών ως φορέων µεταφοράς (Iatrou et al 1989;Iatrou and Meidinger 1990).…”

“…Πράγµατι, γενετικός µετασχηµατισµός της D. melanogaster µε το ζεύγος γονιδίων Α/Β.L12 του B. mori είχε ως αποτέλεσµα την παράλληλη έκφραση των δύο γονιδίων µόνο σε θηλυκά άτοµα και συγκεκριµένα σε ωοθυλάκια που βρίσκονταν στα τελευταία στάδια της χοριογένεσης (Mitsialis and Kafatos 1985). Επίσης, εισαγωγή του ζεύγους γονιδίων HcA/B.12 µέσω του ανασυνδυασµένου ραβδοϊού BmNPV, σε λάρβες του µεταλλαγµένου στελέχους GrB, από το οποίο λείπουν όλα τα Ηc γονίδια (Iatrou et al 1980), οδήγησε στην παραγωγή σωστών HcA και HcB µεταγράφων στα θυλακοκύτταρα των θηλυκών ατόµων (Iatrou and Meidinger 1990). Η λειτουργικότητα της 5' γειτονικής περιοχής αλλά και η ικανότητά της να κατευθύνει ειδικά την έκφραση των γονιδίων και προς τις δύο κατευθύνσεις (αµφίδροµα), επιβεβαιώθηκε για τέσσερα διαφορετικά ζεύγη γονιδίων του χορίου από τρία είδη µεταξοσκώληκα.…”