{"id":{"repo_id":"oxford-brookes","oai_identifier":"tle:88321cec-179e-4218-9524-e7145d60596f:d6bd9758-527a-46cd-bfe2-c433766e8fca:1"},"canonical_url":"https://search.dev.ndltd.org/etd/oxford-brookes/tle:88321cec-179e-4218-9524-e7145d60596f:d6bd9758-527a-46cd-bfe2-c433766e8fca:1","repository":{"repo_id":"oxford-brookes","name":"Oxford Brookes University","base_url":"https://radar.brookes.ac.uk/radar/oai"},"display":{"title":"Gene regulation in baculovirus-infected insect cells : application in the development of a budded virus free expression system","abstract":"The baculovirus expression vector system (BEVS) is a valuable tool for the production of recombinant proteins including complexes such as virus-like particles (VLPs) for vaccine production. A significant limitation of the BEVS, however, is the co-production and subsequent contamination of VLPs and other biopharmaceuticals with the baculovirus budded virus (BV). This is of concern to regulatory bodies as BVs are enriched with the glycoprotein GP64, which is immunogenic, so must be removed from biopharmaceutical preparations. However, the cost of downstream processing to remove these particles is considerable. Consequently, this thesis aimed to develop an upstream solution that inhibits BV production, thus eliminating the need for downstream processing. A novel, selectively repressible system was developed employing components of the Escherichia coli lac operon to block expression of essential genes required for BV production during VLP/protein production (the LacI/O Repressor System). Initially, RNA interference was used in a screening process to inhibit expression of a large number of structural genes associated with BV. Five “key” structural genes (gp64, vp39, Ac83(a), Ac92, and Ac142) were identified as integral to successful assembly and release of BVs, and which could have potential to reduce BV production if repressed by the LacI/O Repressor System. The effects of paired gene silencing by RNAi was also assessed. Data demonstrated that silencing of two “key” genes could further significantly reduce (p<0.05) BV production. For the development of the LacI/O Repressor System, multiple single and dual gene expression promoter constructs (based on the chitinase/cathepsin (chiA/cath) promoter) were produced. Each contained the E. coli lac operator (lacO) at different positions relative to the transcription initiation site (TAAG), and through fluorescence assays and western blotting, their function was confirmed. Basal gene expression from both the single and dual expression chiA/lacO promoters was affected by lacO positioning. Insertion of lacO after the TAAG significantly (p<0.01) increased promoter activity, whilst insertion before the transcription initiation site significantly (p<0.01) decreased activity. Insertion of one lacO after the TAAG motif delayed promoter activity, with the increase in promoter activity due to an increase in promoter strength. Addition of a second lacO after TAAG did not increase promoter activity by further increasing promoter strength. Instead, by extending the length of time the promoter was active. A stable cell line (SfLacI) constitutively producing the E. coli repressor protein, LacI, was established. Infection of these cells with a recombinant virus expressing the “key” genes under the chiA/lacO promoter would allow LacI to bind lacO and block gene expression, resulting in a reduction in BV synthesis. Flow cytometry experiments showed that all single expression chiA/lacO promoter constructs were capable of downregulating gene expression in the presence of LacI. However, only insertion of lacO after the TAAG motif yielded significant levels (p<0.05) of gene inhibition. Increased promoter activity seemed to correlate with greater levels of gene downregulation, which was likely due to the increased rates of RNAP clearance, which increased the opportunity for LacI binding to lacO in the promoter. Addition of a second lacO sequence after the TAAG motif made gene repression more consistent between experimental replicates, possibly by increasing the rate of repressor/operator binding, or by increasing the stability of the repressor-operator complex. The presence of two lacOs spaced further apart enhanced gene repression, possibly by enabling the formation of DNA loops. Significant downregulation of two genes was possible from the dual chiA/lacO promoter construct when in the presence of LacI. When employing the LacI/O Repressor System to downregulate the “key” gene, gp64, BV titres were significantly reduced (p<0.05) by 50%. This is a significant starting point for the development of a BV free expression system and further work is required to enhance the current levels of gene/BV repression.","abstract_html":"The baculovirus expression vector system (BEVS) is a valuable tool for the production of recombinant proteins including complexes such as virus-like particles (VLPs) for vaccine production. A significant limitation of the BEVS, however, is the co-production and subsequent contamination of VLPs and other biopharmaceuticals with the baculovirus budded virus (BV). This is of concern to regulatory bodies as BVs are enriched with the glycoprotein GP64, which is immunogenic, so must be removed from biopharmaceutical preparations. However, the cost of downstream processing to remove these particles is considerable. Consequently, this thesis aimed to develop an upstream solution that inhibits BV production, thus eliminating the need for downstream processing. A novel, selectively repressible system was developed employing components of the Escherichia coli lac operon to block expression of essential genes required for BV production during VLP/protein production (the LacI/O Repressor System). Initially, RNA interference was used in a screening process to inhibit expression of a large number of structural genes associated with BV. Five “key” structural genes (gp64, vp39, Ac83(a), Ac92, and Ac142) were identified as integral to successful assembly and release of BVs, and which could have potential to reduce BV production if repressed by the LacI/O Repressor System. The effects of paired gene silencing by RNAi was also assessed. Data demonstrated that silencing of two “key” genes could further significantly reduce (p&lt;0.05) BV production. For the development of the LacI/O Repressor System, multiple single and dual gene expression promoter constructs (based on the chitinase/cathepsin (chiA/cath) promoter) were produced. Each contained the E. coli lac operator (lacO) at different positions relative to the transcription initiation site (TAAG), and through fluorescence assays and western blotting, their function was confirmed. Basal gene expression from both the single and dual expression chiA/lacO promoters was affected by lacO positioning. Insertion of lacO after the TAAG significantly (p&lt;0.01) increased promoter activity, whilst insertion before the transcription initiation site significantly (p&lt;0.01) decreased activity. Insertion of one lacO after the TAAG motif delayed promoter activity, with the increase in promoter activity due to an increase in promoter strength. Addition of a second lacO after TAAG did not increase promoter activity by further increasing promoter strength. Instead, by extending the length of time the promoter was active. A stable cell line (SfLacI) constitutively producing the E. coli repressor protein, LacI, was established. Infection of these cells with a recombinant virus expressing the “key” genes under the chiA/lacO promoter would allow LacI to bind lacO and block gene expression, resulting in a reduction in BV synthesis. Flow cytometry experiments showed that all single expression chiA/lacO promoter constructs were capable of downregulating gene expression in the presence of LacI. However, only insertion of lacO after the TAAG motif yielded significant levels (p&lt;0.05) of gene inhibition. Increased promoter activity seemed to correlate with greater levels of gene downregulation, which was likely due to the increased rates of RNAP clearance, which increased the opportunity for LacI binding to lacO in the promoter. Addition of a second lacO sequence after the TAAG motif made gene repression more consistent between experimental replicates, possibly by increasing the rate of repressor/operator binding, or by increasing the stability of the repressor-operator complex. The presence of two lacOs spaced further apart enhanced gene repression, possibly by enabling the formation of DNA loops. Significant downregulation of two genes was possible from the dual chiA/lacO promoter construct when in the presence of LacI. When employing the LacI/O Repressor System to downregulate the “key” gene, gp64, BV titres were significantly reduced (p&lt;0.05) by 50%. This is a significant starting point for the development of a BV free expression system and further work is required to enhance the current levels of gene/BV repression.","abstract_has_math":false,"creators":["Backhouse, Victoria"],"institution":"Oxford Brookes University","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":["Possee, Robert","King, Linda"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2022,"date_issued":"2022","date_published":"2022","updated_at":"2026-07-24T03:42:15Z","subjects":[],"languages":["en"],"rights":["All rights reserved"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.24384/b667-3d83","outbound_label":"DOI","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Backhouse, Victoria","Possee, Robert","King, Linda"]},{"key":"dc:creator","label":"Author","values":["Backhouse, Victoria"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2022"]},{"key":"dc:publisher","label":"Institution","values":["Oxford Brookes University"]},{"key":"dc:type","label":"Dc Type","values":["thesis"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["All rights reserved"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://doi.org/10.24384/b667-3d83","https://radar.brookes.ac.uk/radar/file/88321cec-179e-4218-9524-e7145d60596f/1/Backhouse2022GeneRegulation.pdf"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["The baculovirus expression vector system (BEVS) is a valuable tool for the production of recombinant proteins including complexes such as virus-like particles (VLPs) for vaccine production. A significant limitation of the BEVS, however, is the co-production and subsequent contamination of VLPs and other biopharmaceuticals with the baculovirus budded virus (BV). This is of concern to regulatory bodies as BVs are enriched with the glycoprotein GP64, which is immunogenic, so must be removed from biopharmaceutical preparations. However, the cost of downstream processing to remove these particles is considerable. Consequently, this thesis aimed to develop an upstream solution that inhibits BV production, thus eliminating the need for downstream processing. A novel, selectively repressible system was developed employing components of the Escherichia coli lac operon to block expression of essential genes required for BV production during VLP/protein production (the LacI/O Repressor System). Initially, RNA interference was used in a screening process to inhibit expression of a large number of structural genes associated with BV. Five “key” structural genes (gp64, vp39, Ac83(a), Ac92, and Ac142) were identified as integral to successful assembly and release of BVs, and which could have potential to reduce BV production if repressed by the LacI/O Repressor System. The effects of paired gene silencing by RNAi was also assessed. Data demonstrated that silencing of two “key” genes could further significantly reduce (p<0.05) BV production. For the development of the LacI/O Repressor System, multiple single and dual gene expression promoter constructs (based on the chitinase/cathepsin (chiA/cath) promoter) were produced. Each contained the E. coli lac operator (lacO) at different positions relative to the transcription initiation site (TAAG), and through fluorescence assays and western blotting, their function was confirmed. Basal gene expression from both the single and dual expression chiA/lacO promoters was affected by lacO positioning. Insertion of lacO after the TAAG significantly (p<0.01) increased promoter activity, whilst insertion before the transcription initiation site significantly (p<0.01) decreased activity. Insertion of one lacO after the TAAG motif delayed promoter activity, with the increase in promoter activity due to an increase in promoter strength. Addition of a second lacO after TAAG did not increase promoter activity by further increasing promoter strength. Instead, by extending the length of time the promoter was active. A stable cell line (SfLacI) constitutively producing the E. coli repressor protein, LacI, was established. Infection of these cells with a recombinant virus expressing the “key” genes under the chiA/lacO promoter would allow LacI to bind lacO and block gene expression, resulting in a reduction in BV synthesis. Flow cytometry experiments showed that all single expression chiA/lacO promoter constructs were capable of downregulating gene expression in the presence of LacI. However, only insertion of lacO after the TAAG motif yielded significant levels (p<0.05) of gene inhibition. Increased promoter activity seemed to correlate with greater levels of gene downregulation, which was likely due to the increased rates of RNAP clearance, which increased the opportunity for LacI binding to lacO in the promoter. Addition of a second lacO sequence after the TAAG motif made gene repression more consistent between experimental replicates, possibly by increasing the rate of repressor/operator binding, or by increasing the stability of the repressor-operator complex. The presence of two lacOs spaced further apart enhanced gene repression, possibly by enabling the formation of DNA loops. Significant downregulation of two genes was possible from the dual chiA/lacO promoter construct when in the presence of LacI. When employing the LacI/O Repressor System to downregulate the “key” gene, gp64, BV titres were significantly reduced (p<0.05) by 50%. This is a significant starting point for the development of a BV free expression system and further work is required to enhance the current levels of gene/BV repression."]},{"key":"dc:format","label":"Dc Format","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Gene regulation in baculovirus-infected insect cells : application in the development of a budded virus free expression system"]}]}],"canonical_facts":{"dc:contributor":["Backhouse, Victoria","Possee, Robert","King, Linda"],"dc:creator":["Backhouse, Victoria"],"dc:date":["2022"],"dc:description":["The baculovirus expression vector system (BEVS) is a valuable tool for the production of recombinant proteins including complexes such as virus-like particles (VLPs) for vaccine production. A significant limitation of the BEVS, however, is the co-production and subsequent contamination of VLPs and other biopharmaceuticals with the baculovirus budded virus (BV). This is of concern to regulatory bodies as BVs are enriched with the glycoprotein GP64, which is immunogenic, so must be removed from biopharmaceutical preparations. However, the cost of downstream processing to remove these particles is considerable. Consequently, this thesis aimed to develop an upstream solution that inhibits BV production, thus eliminating the need for downstream processing. A novel, selectively repressible system was developed employing components of the Escherichia coli lac operon to block expression of essential genes required for BV production during VLP/protein production (the LacI/O Repressor System). Initially, RNA interference was used in a screening process to inhibit expression of a large number of structural genes associated with BV. Five “key” structural genes (gp64, vp39, Ac83(a), Ac92, and Ac142) were identified as integral to successful assembly and release of BVs, and which could have potential to reduce BV production if repressed by the LacI/O Repressor System. The effects of paired gene silencing by RNAi was also assessed. Data demonstrated that silencing of two “key” genes could further significantly reduce (p<0.05) BV production. For the development of the LacI/O Repressor System, multiple single and dual gene expression promoter constructs (based on the chitinase/cathepsin (chiA/cath) promoter) were produced. Each contained the E. coli lac operator (lacO) at different positions relative to the transcription initiation site (TAAG), and through fluorescence assays and western blotting, their function was confirmed. Basal gene expression from both the single and dual expression chiA/lacO promoters was affected by lacO positioning. Insertion of lacO after the TAAG significantly (p<0.01) increased promoter activity, whilst insertion before the transcription initiation site significantly (p<0.01) decreased activity. Insertion of one lacO after the TAAG motif delayed promoter activity, with the increase in promoter activity due to an increase in promoter strength. Addition of a second lacO after TAAG did not increase promoter activity by further increasing promoter strength. Instead, by extending the length of time the promoter was active. A stable cell line (SfLacI) constitutively producing the E. coli repressor protein, LacI, was established. Infection of these cells with a recombinant virus expressing the “key” genes under the chiA/lacO promoter would allow LacI to bind lacO and block gene expression, resulting in a reduction in BV synthesis. Flow cytometry experiments showed that all single expression chiA/lacO promoter constructs were capable of downregulating gene expression in the presence of LacI. However, only insertion of lacO after the TAAG motif yielded significant levels (p<0.05) of gene inhibition. Increased promoter activity seemed to correlate with greater levels of gene downregulation, which was likely due to the increased rates of RNAP clearance, which increased the opportunity for LacI binding to lacO in the promoter. Addition of a second lacO sequence after the TAAG motif made gene repression more consistent between experimental replicates, possibly by increasing the rate of repressor/operator binding, or by increasing the stability of the repressor-operator complex. The presence of two lacOs spaced further apart enhanced gene repression, possibly by enabling the formation of DNA loops. Significant downregulation of two genes was possible from the dual chiA/lacO promoter construct when in the presence of LacI. When employing the LacI/O Repressor System to downregulate the “key” gene, gp64, BV titres were significantly reduced (p<0.05) by 50%. This is a significant starting point for the development of a BV free expression system and further work is required to enhance the current levels of gene/BV repression."],"dc:format":["application/pdf"],"dc:identifier":["https://doi.org/10.24384/b667-3d83","https://radar.brookes.ac.uk/radar/file/88321cec-179e-4218-9524-e7145d60596f/1/Backhouse2022GeneRegulation.pdf"],"dc:language":["en"],"dc:publisher":["Oxford Brookes University"],"dc:rights":["All rights reserved"],"dc:title":["Gene regulation in baculovirus-infected insect cells : application in the development of a budded virus free expression system"],"dc:type":["thesis"]},"updated_at":"2026-07-24T03:42:15Z"}