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Manipulation of chick embryonic development using retroviral vectors

Bell, Esther Jane de Hauteville; (1994) Manipulation of chick embryonic development using retroviral vectors. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

The aim of this research was to investigate the functions of Retinoic Acid Receptor-β (RAR- β) and Bone Morphogenetic Protein-4 (BMP-4) in pattern formation and morphogenesis in the developing chick wing. The approach was to alter the pattern of RAR-β and BMP-4 expression in wing buds using recombinant retroviruses encoding these proteins. The vector used was RCAS(BP), a replication competent virus derived from Rous Sarcoma Virus. Recombinant viruses were prepared with either human RAR-β or mouse Bmp-4. Both cDNAs encode proteins with a high degree of amino acid sequence identity to their chick counterpart and should function in chicken cells. The constructs were transfected into primary chick embryonic fibroblasts. Ten to fourteen days later the supernatant, which contained infectious virus particles, was harvested. The viral supernatants were then used to infect early chick embryos in either the right wing primordia or the right wing bud in ovo. The infected embryos were analysed for phenotypic changes and for viral spread. Embryos infected with the hRAR-β virus showed very little or no spread of the virus. None of these embryos infected with RCAS(BP) hRAR-β showed any changes in the phenotype. PolyA+ mRNA was extracted from the transfected cells and northern blots were performed. The results showed that splicing of the primary viral RNA to hRAR-β mRNA was very inefficient. In addition, no evidence for expression of hRAR-β could be obtained from a retinoic acid binding assay, a reporter gene assay or from western blotting or immunocytochemistry. In contrast, the RCAS(BP) mBmp-4 virus spread well in the limb bud. Northern blotting showed that Bmp-4 mRNA was efficiently spliced from the primary transcript of the RCAS(BP) mBmp-4 virus. The virus was shown to direct synthesis of functional mBMP-4 by its effects on Xenopus animal caps and on chick limb bud micromass cultures. The RCAS(BP) mBmp-4 virus was then injected into the limb bud, between stages 10 and 26. The results showed that by expressing BMP-4 ectopically in the chick wing, an increase in the width of the cartilage was seen in both the anterior-posterior and dorsal-ventral axes. The number and combination of elements affected varied. This supports the hypothesis that BMP-4 is involved in modelling cartilage formation.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Manipulation of chick embryonic development using retroviral vectors
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
URI: https://discovery.ucl.ac.uk/id/eprint/10101086
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