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Title: Transgenes targeted to growth hormone cells
Author: McGuinness, Lindsay
ISNI:       0000 0001 3624 5892
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 2002
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The relevance of targeting eGFP to GH vesicles is to study a particular dominant-negative GH mutation, which causes dwarfism in children. Green fluorescent proteins (GFPs) are useful in vivo reporter molecules, which can be used to identify and isolate living cells in which they are expressed. I used an expression cassette in which the signal peptide and the first 22 residues of hGH protein were linked in frame with an enhanced GFP (eGFP) sequence. Pituitary GC cells were transfected with a construct linking the GH signal peptide to eGFP and stable fluorescent lines were established. Confocal microscopy confirmed a granular localisation of eGFP. This eGFP reporter cassette was then placed in a cosmid construct containing the locus control region for human GH, and used to generate transgenic mice. Anterior pituitaries from GH-eGFP mice showed numerous clusters of strongly fluorescent somatotrophs; EM showed co-localisation of GFP with granules in pituitary somatotrophs. GH content was lower in pituitaries of transgenic animals compared to wild type litter mates, but this did not appear to impair their growth. GH cells could be purified by FACS. The dominant-negative mutation is an intronic splice site G 'A transition causing exon 3 to be skipped, producing a smaller inactive form of GH (del32-71). The mechanism by which the mutated allele product prevents the release of GH from the normal allele is unknown. Pituitary GC cells were transfected with a hGH gene containing the wild-type hGH or dominant-negative mutation (hGH-IVS3). Stable cell lines were established and hGH-IVS3 transcribed the exon 3 skip mRNA. When co-transfected with GH-eGFP, TIRF (total internal reflection) and confocal microscopy showed a reduction of GFP granules in hGH-IVS3 mutant GC cells and a disruption in cell morphology. RIA of media for rGH from hGH-IVS3 was significantly reduced compared to wild type GC cells. hGH-IVS3 was then placed in the LCR cosmid and expressed in mouse pituitary somatotrophs, and induced profound pituitary GH deficiency and autosomal dominant dwarfism, reduced in weight and length. As expected GH deficiency was associated with an increase in GHRH expression in the arcuate nuclei in these mice. In adult mice from the most severely affected line, the anterior pituitary glands were profoundly hypoplastic with few recognizable somatotrophs, and their morphology showed a similar granular disruption seen in cell lines with evident macrophage invasion. These transgenic models provide us with the means to study the development and the progression of the pathology of human dominant-negative dwarfism in a rodent model in vivo.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available