Richard Halley-Stott | University of Cape Town (original) (raw)
Papers by Richard Halley-Stott
Trends in Genetics, Dec 1, 2011
Transcription, 2015
N uclear reprogramming changes the identity of cells by changing gene expression programs. Two re... more N uclear reprogramming changes the identity of cells by changing gene expression programs. Two recent pieces of work have highlighted the role that mitosis plays in enhancing the success of nuclear reprogramming. This Point of View article examines this work in the context of nuclear reprogramming.
Journal of Cell Science, Dec 15, 2012
How cell fate becomes restricted during somatic cell differentiation is a long-lasting question i... more How cell fate becomes restricted during somatic cell differentiation is a long-lasting question in biology. Epigenetic mechanisms not present in pluripotent cells and acquired during embryonic development are expected to stabilize the differentiated state of somatic cells and thereby restrict their ability to convert to another fate. The histone variant macroH2A acts as a component of an epigenetic multilayer that heritably maintains the silent X chromosome and has been shown to restrict tumor development. Here we show that macroH2A marks the differentiated cell state during mouse embryogenesis. MacroH2A.1 was found to be present at low levels upon the establishment of pluripotency in the inner cell mass and epiblast, but it was highly enriched in the trophectoderm and differentiated somatic cells later in mouse development. Chromatin immunoprecipitation revealed that macroH2A.1 is incorporated in the chromatin of regulatory regions of pluripotency genes in somatic cells such as mouse embryonic fibroblasts and adult neural stem cells, but not in embryonic stem cells. Removal of macroH2A.1, macroH2A.2 or both increased the efficiency of induced pluripotency up to 25-fold. The obtained induced pluripotent stem cells reactivated pluripotency genes, silenced retroviral transgenes and contributed to chimeras. In addition, overexpression of macroH2A isoforms prevented efficient reprogramming of epiblast stem cells to naïve pluripotency. In summary, our study identifies for the first time a link between an epigenetic mark and cell fate restriction during somatic cell differentiation, which helps to maintain cell identity and antagonizes induction of a pluripotent stem cell state.
Briefings in Functional Genomics, Apr 12, 2013
Epigenetic memory represents a natural mechanism whereby the identity of a cell is maintained thr... more Epigenetic memory represents a natural mechanism whereby the identity of a cell is maintained through successive cell cycles, allowing the specification and maintenance of differentiation during development and in adult cells. Cancer is a loss or reversal of the stable differentiated state of adult cells and may be mediated in part by epigenetic changes. The identity of somatic cells can also be reversed experimentally by nuclear reprogramming. Nuclear reprogramming experiments reveal the mechanisms required to activate embryonic gene expression in adult cells and thus provide insight into the reversal of epigenetic memory. In this article, we will introduce epigenetic memory and the mechanisms by which it may operate. We limit our discussion primarily to the context of nuclear reprogramming and briefly discuss the relevance of memory and reprogramming to cancer biology.
Communicative & Integrative Biology, Jul 1, 2012
South African Medical Journal, Jul 26, 2019
This open-access article is distributed under Creative Commons licence CC-BY-NC 4.0.
Archives of Virology, Mar 9, 2007
PLOS Biology, Jul 29, 2014
Cell differentiation is remarkably stable but can be reversed by somatic cell nuclear transfer, c... more Cell differentiation is remarkably stable but can be reversed by somatic cell nuclear transfer, cell fusion, and iPS. Nuclear transfer to amphibian oocytes provides a special opportunity to test transcriptional reprogramming without cell division. We show here that, after nuclear transfer to amphibian oocytes, mitotic chromatin is reprogrammed up to 100 times faster than interphase nuclei. We find that, as cells traverse mitosis, their genes pass through a temporary phase of unusually high responsiveness to oocyte reprogramming factors (mitotic advantage). Mitotic advantage is not explained by nuclear penetration, DNA modifications, histone acetylation, phosphorylation, methylation, nor by salt soluble chromosomal proteins. Our results suggest that histone H2A deubiquitination may account, at least in part, for the acquisition of mitotic advantage. They support the general principle that a temporary access of cytoplasmic factors to genes during mitosis may facilitate somatic cell nuclear reprogramming and the acquisition of new cell fates in normal development.
Plant Biotechnology Journal, 2010
We constructed a novel autonomously replicating gene expression shuttle vector, with the aim of d... more We constructed a novel autonomously replicating gene expression shuttle vector, with the aim of developing a system for transiently expressing proteins at levels useful for commercial production of vaccines and other proteins in plants. The vector, pRIC, is based on the mild strain of the geminivirus Bean yellow dwarf virus (BeYDVm) and is replicationally released into plant cells from a recombinant Agrobacterium tumefaciens Ti plasmid. pRIC differs from most other geminivirus-based vectors in that the BeYDV replication-associated elements were included in cis rather than from a co-transfected plasmid, while the BeYDV capsid protein (CP) and movement protein (MP) genes were replaced by an antigen encoding transgene expression cassette derived from the non-replicating A. tumefaciens vector, pTRAc. We tested vector efficacy in Nicotiana benthamiana by comparing transient cytoplasmic expression between pRIC and pTRAc constructs encoding either enhanced green fluorescent protein (EGFP) or the subunit vaccine antigens, human papillomavirus subtype 16 (HPV-16) major CP L1 and human immunodeficiency virus subtype C p24 antigen. The pRIC constructs were amplified in planta by up to two orders of magnitude by replication, while 50% more HPV-16 L1 and three-to seven-fold more EGFP and HIV-1 p24 were expressed from pRIC than from pTRAc. Vector replication was shown to be correlated with increased protein expression. We anticipate that this new high-yielding plant expression vector will contribute towards the development of a viable plant production platform for vaccine candidates and other pharmaceuticals.
Development, Jun 15, 2013
There is currently particular interest in the field of nuclear reprogramming, a process by which ... more There is currently particular interest in the field of nuclear reprogramming, a process by which the identity of specialised cells may be changed, typically to an embryonic-like state. Reprogramming procedures provide insight into many mechanisms of fundamental cell biology and have several promising applications, most notably in healthcare through the development of human disease models and patient-specific tissue-replacement therapies. Here, we introduce the field of nuclear reprogramming and briefly discuss six of the procedures by which reprogramming may be experimentally performed: nuclear transfer to eggs or oocytes, cell fusion, extract treatment, direct reprogramming to pluripotency and transdifferentiation.
South African Medical Journal, 2019
This open-access article is distributed under Creative Commons licence CC-BY-NC 4.0.
BMJ Global Health, 2021
BackgroundGlobal surgery has recently gained prominence as an academic discipline within global h... more BackgroundGlobal surgery has recently gained prominence as an academic discipline within global health. Authorship inequity has been a consistent feature of global health publications, with over-representation of authors from high-income countries (HICs), and disenfranchisement of researchers from low-income and middle-income countries (LMICs). In this study, we investigated authorship demographics within recently published global surgery literature.MethodsWe performed a systematic analysis of author characteristics, including gender, seniority and institutional affiliation, for global surgery studies published between 2016 and 2020 and indexed in the PubMed database. We compared the distribution of author gender and seniority across studies related to different topics; between authors affiliated with HICs and LMICs; and across studies with different authorship networks.Results1240 articles were included for analysis. Most authors were male (60%), affiliated only with HICs (51%) and...
memory in the context of nuclear reprogramming and cancer
Bean yellow dwarf virus (BeYDV) is a single-stranded DNA virus in the genus Mastrevirus [8] ofthe... more Bean yellow dwarf virus (BeYDV) is a single-stranded DNA virus in the genus Mastrevirus [8] ofthe family Geminiviridae [14]. The majority of mas-treviruses infect monocotyledonous hosts, specif-ically members of the Poaceae (Gramineae). Onlytwo confirmed mastreviruses infect dicotyledonousplants: tobacco yellow dwarf virus [11] and BeYDV[8]. Chickpea chlorotic dwarf virus (CpCDV) hasbeen tentatively assigned as a third mastrevirus thatinfects dicotyledonous hosts [14]. Mastreviruses aretransmitted in nature by leafhoppers (Homoptera,Cicadelidae), and in most cases a single mastrevirusspecies is transmitted by a single leafhopper species.To date, only a single example of BeYDV has beendescribed, and its insect vector is unknown [8]. Here,we report on the nucleotide sequence of a second,phenotypicallydistinct BeYDV strain, which wepro-pose calling BeYDV-mild (BeYDV-m).The BeYDV-m genome was isolated from nat-urally infected bean plants (Phaseolus vulgariscv. Top Crop) obtained via the ...
British Medical Journal Global Health, 2021
Background Global surgery has recently gained prominence as an academic discipline within global ... more Background Global surgery has recently gained prominence as an academic discipline within global health. Authorship inequity has been a consistent feature of global health publications, with over-representation of authors from high-income countries (HICs), and disenfranchisement of researchers from low-income and middle-income countries (LMICs). In this study, we investigated authorship demographics within recently published global surgery literature. Methods We performed a systematic analysis of author characteristics, including gender, seniority and institutional affiliation, for global surgery studies published between 2016 and 2020 and indexed in the PubMed database. We compared the distribution of author gender and seniority across studies related to different topics; between authors affiliated with HICs and LMICs; and across studies with different authorship networks. Results 1240 articles were included for analysis. Most authors were male (60%), affiliated only with HICs (51%) and of high seniority (55% were fully qualified specialist or generalist clinicians, Principal Investigators, or in senior leadership or management roles). The proportion of male authors increased with increasing seniority for last and middle authors. Studies related to Obstetrics and Gynaecology had similar numbers of male and female authors, whereas there were more male authors in studies related to surgery (69% male) and Anaesthesia and Critical care (65% male). Compared with HIC authors, LMIC authors had a lower proportion of female authors at every seniority grade. This gender gap among LMIC middle authors was reduced in studies where all authors were affiliated only with LMICs. Conclusion Authorship disparities are evident within global surgery academia. Remedial actions to address the lack of authorship opportunities for LMIC authors and female authors are required.
Stem Cell Reviews and Reports
The Primary Scarring Alopecias are characterised by the irreversible destruction and fibrosis of ... more The Primary Scarring Alopecias are characterised by the irreversible destruction and fibrosis of hair follicles, leading to permanent and often disfiguring loss of hair. The pathophysiology of these diseases is not well understood. However, follicular-fibrosis and loss of the stem-cell niche appears to be a common theme. This review explores the pathogenesis of primary scarring alopecias, asking what happens to the stem cells of the hair follicle and how they may contribute to the progression of these diseases. Bulge-resident cells are lost (leading to loss of capacity for hair growth) from the follicle either by inflammatory-mediate apoptosis or through epigenetic reprogramming to assume a mesenchymal-like identity. What proportion of bulge cells is lost to which process is unknown and probably differs depending on the individual PCA and its specific inflammatory cell infiltrate. The formation of fibroblast-like cells from follicular stem cells may also mean that the cells of the bulge have a direct role in the pathogenesis. The identification of specific cells involved in the pathogenesis of these diseases could provide unique diagnostic and therapeutic opportunities to prevent disease progression by preventing EMT and specific pro-fibrotic signals.
The high cost of new generation vaccines makes them largely inaccessible to developing countries.... more The high cost of new generation vaccines makes them largely inaccessible to developing countries. Production of vaccines in plants offers a low cost alternative, but is currently hampered by low expression levels of recombinant protein. A novel solution to this problem is to develop plant expression vectors capable of transgene copy number amplification. We used genomic sequence from Bean yellow dwarf virus (BeYDV) to construct an autonomously replicating plant expression vector, pRIC; which includes a transgene expression cassette within a replicon that is designed to undergo replicational release in planta from an Agrobacterium tumefaciens binary vector. Subsequent vector amplification would lead to amplified transgene copy number. We compared pRIC functionality in Nicotiana benthamiana against that of a non-replicating plant vector, pTRAc. Replicational release of pRIC was investigated using quantitative PCR. Western blot analysis, ELISA and quantitative analysis of EGFP fluorescence intensity were used, as appropriate, to test expression of three reporter genes cloned into each vector: human codon optimised Human Papillomavirus (HPV) L1 coat protein, enhanced green fluorescent protein (EGFP) and HIV-1C p24. Replicational release and vector amplification of pRIC were demonstrated to occur in host plants, indicating that pRIC functioned as designed. Reporter proteins were expressed at higher levels in plants transfected with pRIC than those transfected with the non-replicating vector pTRAc; for example, expression of HPV L1 protein was ten-fold higher in pRIC – transfected plants than in pTRAc – transfected plants. The BeYDV-based plant expression vector, pRIC, was shown to replicate in N. benthamiana, leading to increased expression of recombinant protein. Thus, amplification of heterologous genes through the use of replicating vectors provides a useful option for increasing vaccinogen expression in plants.
Trends in Genetics, Dec 1, 2011
Transcription, 2015
N uclear reprogramming changes the identity of cells by changing gene expression programs. Two re... more N uclear reprogramming changes the identity of cells by changing gene expression programs. Two recent pieces of work have highlighted the role that mitosis plays in enhancing the success of nuclear reprogramming. This Point of View article examines this work in the context of nuclear reprogramming.
Journal of Cell Science, Dec 15, 2012
How cell fate becomes restricted during somatic cell differentiation is a long-lasting question i... more How cell fate becomes restricted during somatic cell differentiation is a long-lasting question in biology. Epigenetic mechanisms not present in pluripotent cells and acquired during embryonic development are expected to stabilize the differentiated state of somatic cells and thereby restrict their ability to convert to another fate. The histone variant macroH2A acts as a component of an epigenetic multilayer that heritably maintains the silent X chromosome and has been shown to restrict tumor development. Here we show that macroH2A marks the differentiated cell state during mouse embryogenesis. MacroH2A.1 was found to be present at low levels upon the establishment of pluripotency in the inner cell mass and epiblast, but it was highly enriched in the trophectoderm and differentiated somatic cells later in mouse development. Chromatin immunoprecipitation revealed that macroH2A.1 is incorporated in the chromatin of regulatory regions of pluripotency genes in somatic cells such as mouse embryonic fibroblasts and adult neural stem cells, but not in embryonic stem cells. Removal of macroH2A.1, macroH2A.2 or both increased the efficiency of induced pluripotency up to 25-fold. The obtained induced pluripotent stem cells reactivated pluripotency genes, silenced retroviral transgenes and contributed to chimeras. In addition, overexpression of macroH2A isoforms prevented efficient reprogramming of epiblast stem cells to naïve pluripotency. In summary, our study identifies for the first time a link between an epigenetic mark and cell fate restriction during somatic cell differentiation, which helps to maintain cell identity and antagonizes induction of a pluripotent stem cell state.
Briefings in Functional Genomics, Apr 12, 2013
Epigenetic memory represents a natural mechanism whereby the identity of a cell is maintained thr... more Epigenetic memory represents a natural mechanism whereby the identity of a cell is maintained through successive cell cycles, allowing the specification and maintenance of differentiation during development and in adult cells. Cancer is a loss or reversal of the stable differentiated state of adult cells and may be mediated in part by epigenetic changes. The identity of somatic cells can also be reversed experimentally by nuclear reprogramming. Nuclear reprogramming experiments reveal the mechanisms required to activate embryonic gene expression in adult cells and thus provide insight into the reversal of epigenetic memory. In this article, we will introduce epigenetic memory and the mechanisms by which it may operate. We limit our discussion primarily to the context of nuclear reprogramming and briefly discuss the relevance of memory and reprogramming to cancer biology.
Communicative & Integrative Biology, Jul 1, 2012
South African Medical Journal, Jul 26, 2019
This open-access article is distributed under Creative Commons licence CC-BY-NC 4.0.
Archives of Virology, Mar 9, 2007
PLOS Biology, Jul 29, 2014
Cell differentiation is remarkably stable but can be reversed by somatic cell nuclear transfer, c... more Cell differentiation is remarkably stable but can be reversed by somatic cell nuclear transfer, cell fusion, and iPS. Nuclear transfer to amphibian oocytes provides a special opportunity to test transcriptional reprogramming without cell division. We show here that, after nuclear transfer to amphibian oocytes, mitotic chromatin is reprogrammed up to 100 times faster than interphase nuclei. We find that, as cells traverse mitosis, their genes pass through a temporary phase of unusually high responsiveness to oocyte reprogramming factors (mitotic advantage). Mitotic advantage is not explained by nuclear penetration, DNA modifications, histone acetylation, phosphorylation, methylation, nor by salt soluble chromosomal proteins. Our results suggest that histone H2A deubiquitination may account, at least in part, for the acquisition of mitotic advantage. They support the general principle that a temporary access of cytoplasmic factors to genes during mitosis may facilitate somatic cell nuclear reprogramming and the acquisition of new cell fates in normal development.
Plant Biotechnology Journal, 2010
We constructed a novel autonomously replicating gene expression shuttle vector, with the aim of d... more We constructed a novel autonomously replicating gene expression shuttle vector, with the aim of developing a system for transiently expressing proteins at levels useful for commercial production of vaccines and other proteins in plants. The vector, pRIC, is based on the mild strain of the geminivirus Bean yellow dwarf virus (BeYDVm) and is replicationally released into plant cells from a recombinant Agrobacterium tumefaciens Ti plasmid. pRIC differs from most other geminivirus-based vectors in that the BeYDV replication-associated elements were included in cis rather than from a co-transfected plasmid, while the BeYDV capsid protein (CP) and movement protein (MP) genes were replaced by an antigen encoding transgene expression cassette derived from the non-replicating A. tumefaciens vector, pTRAc. We tested vector efficacy in Nicotiana benthamiana by comparing transient cytoplasmic expression between pRIC and pTRAc constructs encoding either enhanced green fluorescent protein (EGFP) or the subunit vaccine antigens, human papillomavirus subtype 16 (HPV-16) major CP L1 and human immunodeficiency virus subtype C p24 antigen. The pRIC constructs were amplified in planta by up to two orders of magnitude by replication, while 50% more HPV-16 L1 and three-to seven-fold more EGFP and HIV-1 p24 were expressed from pRIC than from pTRAc. Vector replication was shown to be correlated with increased protein expression. We anticipate that this new high-yielding plant expression vector will contribute towards the development of a viable plant production platform for vaccine candidates and other pharmaceuticals.
Development, Jun 15, 2013
There is currently particular interest in the field of nuclear reprogramming, a process by which ... more There is currently particular interest in the field of nuclear reprogramming, a process by which the identity of specialised cells may be changed, typically to an embryonic-like state. Reprogramming procedures provide insight into many mechanisms of fundamental cell biology and have several promising applications, most notably in healthcare through the development of human disease models and patient-specific tissue-replacement therapies. Here, we introduce the field of nuclear reprogramming and briefly discuss six of the procedures by which reprogramming may be experimentally performed: nuclear transfer to eggs or oocytes, cell fusion, extract treatment, direct reprogramming to pluripotency and transdifferentiation.
South African Medical Journal, 2019
This open-access article is distributed under Creative Commons licence CC-BY-NC 4.0.
BMJ Global Health, 2021
BackgroundGlobal surgery has recently gained prominence as an academic discipline within global h... more BackgroundGlobal surgery has recently gained prominence as an academic discipline within global health. Authorship inequity has been a consistent feature of global health publications, with over-representation of authors from high-income countries (HICs), and disenfranchisement of researchers from low-income and middle-income countries (LMICs). In this study, we investigated authorship demographics within recently published global surgery literature.MethodsWe performed a systematic analysis of author characteristics, including gender, seniority and institutional affiliation, for global surgery studies published between 2016 and 2020 and indexed in the PubMed database. We compared the distribution of author gender and seniority across studies related to different topics; between authors affiliated with HICs and LMICs; and across studies with different authorship networks.Results1240 articles were included for analysis. Most authors were male (60%), affiliated only with HICs (51%) and...
memory in the context of nuclear reprogramming and cancer
Bean yellow dwarf virus (BeYDV) is a single-stranded DNA virus in the genus Mastrevirus [8] ofthe... more Bean yellow dwarf virus (BeYDV) is a single-stranded DNA virus in the genus Mastrevirus [8] ofthe family Geminiviridae [14]. The majority of mas-treviruses infect monocotyledonous hosts, specif-ically members of the Poaceae (Gramineae). Onlytwo confirmed mastreviruses infect dicotyledonousplants: tobacco yellow dwarf virus [11] and BeYDV[8]. Chickpea chlorotic dwarf virus (CpCDV) hasbeen tentatively assigned as a third mastrevirus thatinfects dicotyledonous hosts [14]. Mastreviruses aretransmitted in nature by leafhoppers (Homoptera,Cicadelidae), and in most cases a single mastrevirusspecies is transmitted by a single leafhopper species.To date, only a single example of BeYDV has beendescribed, and its insect vector is unknown [8]. Here,we report on the nucleotide sequence of a second,phenotypicallydistinct BeYDV strain, which wepro-pose calling BeYDV-mild (BeYDV-m).The BeYDV-m genome was isolated from nat-urally infected bean plants (Phaseolus vulgariscv. Top Crop) obtained via the ...
British Medical Journal Global Health, 2021
Background Global surgery has recently gained prominence as an academic discipline within global ... more Background Global surgery has recently gained prominence as an academic discipline within global health. Authorship inequity has been a consistent feature of global health publications, with over-representation of authors from high-income countries (HICs), and disenfranchisement of researchers from low-income and middle-income countries (LMICs). In this study, we investigated authorship demographics within recently published global surgery literature. Methods We performed a systematic analysis of author characteristics, including gender, seniority and institutional affiliation, for global surgery studies published between 2016 and 2020 and indexed in the PubMed database. We compared the distribution of author gender and seniority across studies related to different topics; between authors affiliated with HICs and LMICs; and across studies with different authorship networks. Results 1240 articles were included for analysis. Most authors were male (60%), affiliated only with HICs (51%) and of high seniority (55% were fully qualified specialist or generalist clinicians, Principal Investigators, or in senior leadership or management roles). The proportion of male authors increased with increasing seniority for last and middle authors. Studies related to Obstetrics and Gynaecology had similar numbers of male and female authors, whereas there were more male authors in studies related to surgery (69% male) and Anaesthesia and Critical care (65% male). Compared with HIC authors, LMIC authors had a lower proportion of female authors at every seniority grade. This gender gap among LMIC middle authors was reduced in studies where all authors were affiliated only with LMICs. Conclusion Authorship disparities are evident within global surgery academia. Remedial actions to address the lack of authorship opportunities for LMIC authors and female authors are required.
Stem Cell Reviews and Reports
The Primary Scarring Alopecias are characterised by the irreversible destruction and fibrosis of ... more The Primary Scarring Alopecias are characterised by the irreversible destruction and fibrosis of hair follicles, leading to permanent and often disfiguring loss of hair. The pathophysiology of these diseases is not well understood. However, follicular-fibrosis and loss of the stem-cell niche appears to be a common theme. This review explores the pathogenesis of primary scarring alopecias, asking what happens to the stem cells of the hair follicle and how they may contribute to the progression of these diseases. Bulge-resident cells are lost (leading to loss of capacity for hair growth) from the follicle either by inflammatory-mediate apoptosis or through epigenetic reprogramming to assume a mesenchymal-like identity. What proportion of bulge cells is lost to which process is unknown and probably differs depending on the individual PCA and its specific inflammatory cell infiltrate. The formation of fibroblast-like cells from follicular stem cells may also mean that the cells of the bulge have a direct role in the pathogenesis. The identification of specific cells involved in the pathogenesis of these diseases could provide unique diagnostic and therapeutic opportunities to prevent disease progression by preventing EMT and specific pro-fibrotic signals.
The high cost of new generation vaccines makes them largely inaccessible to developing countries.... more The high cost of new generation vaccines makes them largely inaccessible to developing countries. Production of vaccines in plants offers a low cost alternative, but is currently hampered by low expression levels of recombinant protein. A novel solution to this problem is to develop plant expression vectors capable of transgene copy number amplification. We used genomic sequence from Bean yellow dwarf virus (BeYDV) to construct an autonomously replicating plant expression vector, pRIC; which includes a transgene expression cassette within a replicon that is designed to undergo replicational release in planta from an Agrobacterium tumefaciens binary vector. Subsequent vector amplification would lead to amplified transgene copy number. We compared pRIC functionality in Nicotiana benthamiana against that of a non-replicating plant vector, pTRAc. Replicational release of pRIC was investigated using quantitative PCR. Western blot analysis, ELISA and quantitative analysis of EGFP fluorescence intensity were used, as appropriate, to test expression of three reporter genes cloned into each vector: human codon optimised Human Papillomavirus (HPV) L1 coat protein, enhanced green fluorescent protein (EGFP) and HIV-1C p24. Replicational release and vector amplification of pRIC were demonstrated to occur in host plants, indicating that pRIC functioned as designed. Reporter proteins were expressed at higher levels in plants transfected with pRIC than those transfected with the non-replicating vector pTRAc; for example, expression of HPV L1 protein was ten-fold higher in pRIC – transfected plants than in pTRAc – transfected plants. The BeYDV-based plant expression vector, pRIC, was shown to replicate in N. benthamiana, leading to increased expression of recombinant protein. Thus, amplification of heterologous genes through the use of replicating vectors provides a useful option for increasing vaccinogen expression in plants.