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Title: Targeted gene integration for the production of recombinant pharmaceuticals in plants
Author: Collison, Sean Alfred
ISNI:       0000 0004 6057 6562
Awarding Body: St George's, University of London
Current Institution: St George's, University of London
Date of Award: 2016
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Production of recombinant protein pharmaceuticals in plants is to become an important biotechnology for improving global health. In addition to having particular advantages for responding to pandemics it also addresses the need for affordable production of pharmaceuticals in developing countries. Whilst the development of transient expression systems has greatly improved the issue of recombinant protein yield from plants, improved expression vectors have also enhanced production from transgenic plants, and there are still drug products for which the technical simplicity of transgenic plant manufacture is important. For transgenic approaches, transgene insertion characteristics such as copy number and sites of insertion are known to affect transgene expression, so the random nature of transgenesis using agrobacterium is problematic, requiring massive screening campaigns to identify optimal lead plant lines. Furthermore, regulatory bodies require detailed characterisation of all genomic alterations resulting from the transformation process. Screening plant lines and genomic and phenotypic analysis is a significant time and cost burden. This thesis describes the design and construction of a series of vectors to develop a targeted gene integration TGI system, utilising the site-specific recombinases PhiC31 integrase and Cre to facilitate predictable gene expression and simplify genomic characterisation by inserting transgenes into predetermined target sites. PhiC31 was applied to perform recombinase- mediated cassette exchange (RMCE), using a promoter-trap system for selection for recombination of the incoming cassette in the correct orientation and allowing the generation of marker-free plants. Cre allowed potential excision of any ectopic integration events occurring during agrobacterium transformation. Over 89 TO target plant lines were generated and screened for recombinant protein production of the fluorescent marker protein, dsRED. They were self-fertilised and 43 T1 progeny were screened by segregation on selective medium. Expression analysis indicated higher transgene expression was associated with single-locus T-DNA insertion. The variation in dsRED expression at TO was also seen in T1 target plant lines, indicating that transgene integration sites are a determining factor for expression yields. Genomic analysis of 22 selected target lines was performed by Southern blot. Single copy T1 target plant lines were transformed with vectors for recombinases and an integration cassette by a novel strategy, to perform RMCE. Putative integrated plant lines were selected for the RMCE event. In a small number of regenerants, loss of dsRED and gain of GUS expression was observed at TO and subsequently in TO backcross plants, indicating successful exchange of the DNA cassettes. This thesis paves the way for a predictable and rapid approach to develop stable transgenic tobacco plants for the production of recombinant pharmaceutical proteins.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available