Monica Bergem disputerte 12. mai 2006

Ane Skjelfjord

Honningbien er blant verdens kommersielt sett viktigste insekter. Avl har foregått på tradisjonelt vis, ved paring av individer med attraktive egenskaper. De siste årene har imidlertid teknologien gjort det mulig å klone husdyr. I 2005 ble bananfluen klonet, som det første insektet i historien.

Advances in methodology for reproductive cloning and developmental studies of the honeybee (Apis mellifera)

dfs

English language summary
The honeybee (Apis mellifera) is one of the main commercially important insect species due to its role as producer of honey and beeswax as well as being one of the major pollinators in agriculture. Recently, the honeybee has gained increasing interest as a model organism within research areas such as aging, social behaviour, social evolution and neurobiology. The value of maintaining genetically favourable lines by use of various breeding strategies is indisputable, and thus reproductive cloning has become, as for a long range of domestic animals, one of the strategies to achieve this.

The establishment of a cloning technology requires the development of intricate methodology including nuclear transfer, cell transplantation, enucleation and maintenance of cells in vitro. Such methodology is based on thorough knowledge of early developmental processes of the species in question. Honeybee embryogenesis has been object to considerable studies, which has resulted in a well-defined understanding of the early processes in the honeybee egg. In this study we utilized established knowledge to develop methodology for reproductive cloning and developmental studies of honeybees.

The first paper in this thesis, describes the development of procedures for transfer of cells from pre-gastrula stage donor embryos (32-34 hours after egg laying) into recipient embryos to create chimeras; individuals with mixed parental lineages. The ability to perform transplantation of cells into honeybee embryos represents an advance in the development of a cloning technology. In addition, this methodology is an advantage for developmental studies by providing novel opportunities for studies of chimerism in bees, which for other species have proved to be highly instrumental in providing insights into e.g. cell fates, differentiation and developmental potential.

Following nuclear transfers and cell transplantations, it is essential to have accurate tools to detect individuals with successfully incorporated donor nuclei or cells. In our first paper, we used microsatellite analysis to detect chimeras, including fluorescently labelled primers for PCR, microcapillary electrophoresis and automated quantification. In our second paper we employed Quantitative Fluorescent PCR (QF-PCR) on microsatellite, and standard curves, to obtain higher accuracy in determination of the genetic composition of bee larvae after cell transplantations. By use of this method we were able to quantify the percentage of donor-recipient genotype ratios, with 1% sensitivity. This method may be highly valuable for studies where activation and reprogramming of cells injected into embryos are investigated.

Continuous cell cultures for honeybees have proved to be challenging and no previous study has, to our knowledge, been able to rear cells for more than a month. Included in a methodological basis for reproductive cloning, access to long-term cell cultures is essential and closely related to the availability of donor nuclei and cells for transplantations. In addition, in vitro cultivation of cells allows for exploration of in vivo mechanisms that are otherwise unachievable.

In our third paper, we initiated more than 150 cultures of embryo cells under various growth conditions. Pre-gastrula stage embryos were found to be most favourable for initiation of cell cultures, and cells were reared successfully in Grace insect medium supplemented with 15% Foetal Bovine Serum. Under these conditions, embryonic cells proliferated in clusters and maintained viable and mitotic for more than three months. Our results constitute the first report on successful long-term cultivation of honeybee cells, which is a valuable tool with applications within reproductive cloning, developmental biology as well as additional research areas.

Pathology is one of the research fields that may benefit from the ability to rear honeybee cells in vitro. In our fourth paper we infected embryonic cell cultures with deformed wing virus (DWV), a honeybee-specific RNA virus, known to cause deformities and death in honeybee pupae. We studied the ability of this virus to propagate (assayed by ELISA) and to cause cytopathic effects in cultivated embryonic cells. However, no traceable signs of replication were found, which suggest that the virus is host specific, and only replicates in cells of pupae.

Key words/ISI database key words: picorna-like virus; drosophila-melanogaster; insect cells; cell lines; bee; hymenoptera; cell transplantation; nuclear transfer; genetic markers; microsatellites; chimeras; ; chimerism; germ-line chimeras; injection; eggs; embryo; blastocyst; nuclei; development; life; Quantitative Fluorescent PCR (QF-PCR); honeybee; Apis mellifera; reproductive cloning; embryo cloning

Nøkkelord til artikkelsøk: insekt, honningbie, bie, Apis mellifera, kloning, reproduktiv kloning, drosophila-melanogaster, celle, cellekultur, hymenoptera, celle transplantasjon, genetiske markører, mikrosatellitter, egg, embryo, blastocyst, liv, utvikling, kimær, mosaikkdyr

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Oppdatert: 21.09.06
Utskriftsvennlig versjon : Arkiv : Tips en venn